JP2013534812A - Fibronectin based scaffold proteins with improved stability - Google Patents

Abstract

  The present application provides fibronectin based scaffold proteins that are associated with improved stability. The application also relates to stable formulations of fibronectin based scaffold proteins and their use in diagnostic, research and therapeutic applications. The present application further relates to cells containing such proteins, polynucleotides encoding such proteins, or fragments thereof, and vectors containing such polynucleotides.

Description

RELATED APPLICATIONS This application is filed on May 26, 2010 and US Provisional Patent Application No. 61 / 348,647, filed May 26, 2010, which is incorporated herein by reference in its entirety. Insist on the benefits of 61 / 348,663.

Introduction Fibronectin-based scaffolds are a family of proteins that can be evolved to bind to any compound of interest. In general, these proteins using scaffolds derived from fibronectin type III (Fn3) or Fn3-like domains are characterized by natural or engineered antibodies (ie polyclonal, monoclonal or single chain antibodies). Functions and has structural advantages. Specifically, the structure of these antibody mimetics is designed to optimize folding, stability and solubility, even under conditions that usually lead to loss of structure and function in the antibody. An example of a fibronectin based scaffold protein is the Adnectin ^trademark (Adnexus, a wholly owned subsidiary of Bristol-Myers Squibb).

  Fibronectin is a large protein that plays an important role in extracellular matrix formation and cell-cell interactions; it consists of multiple repeats of three (I, II and III) small domains (Baron et al.,). 1991). Fn3 itself is a large subfamily paradigm that includes parts of cell adhesion molecules, cell surface hormones and cytokine receptors, chaperones and carbohydrate binding domains. For reviews, see Bork & Doolittle, Proc Natl Acad Sci USA. 1992 Oct 1; 89 (19): 8990-4; Bork et al. , J Mol Biol. 1994 Sep 30; 242 (4): 309-20; Campbell & Spitzfaden, Structure. 1994 May 15; 2 (5): 333-7; Harpez & Chothia, J Mol Biol. 1994 May 13; 238 (4): 528-39).

  The fibronectin type III (Fn3) domain is, in order from N-terminal to C-terminal, β or β-like chain, A; loop, AB; β or β-like chain, B; loop, BC; β or β-like chain, C; , CD; β or β-like chain, D; loop, DE; β or β-like chain, E; loop, EF; β or β-like chain, F; loop, FG; and β or β-like chain, G. Any or all of loops AB, BC, CD, DE, EF and FG may be involved in target binding. The BC, DE, and FG loops are structurally and functionally similar to complementarity determining regions (CDRs) from immunoglobulins. US Pat. No. 7,115,396 describes Fn3 domain proteins in which changes in the BC, DE and FG loops result in high affinity TNFα binders. US Publication No. 2007/0148126 describes Fn3 domain proteins in which changes in BC, DE and FG loops result in high affinity VEGFR2 binders.

  Protein formulations may involve physical and chemical instabilities during their production, purification, storage and transportation. These instability problems adversely affect the biological properties associated with protein therapy, thereby reducing the effectiveness of the protein therapy. Sola, 2009, J.A. Pharm Sci, 98 (4): 1223-1245. Thus, obtaining an improved fibronectin domain scaffold protein that is associated with improved stability, eg, reduced fragmentation and / or aggregation, and can be used for both therapeutic and diagnostic purposes. Would be advantageous.

SUMMARY One aspect of the present application provides novel fibronectin based scaffold proteins that are associated with increased stability, including reduced fragmentation and / or reduced aggregation.

In some embodiments, a fibronectin based scaffold protein provided herein comprises a fibronectin type III 10 ( ¹⁰ Fn3) domain, wherein the ¹⁰ Fn3 domain comprises SEQ ID NO: 1 and It comprises an amino acid sequence having at least 60% identity are those that bind to a target molecule with a _{K D} of less than 100 nM, and the ¹⁰ Fn3 domain are those further comprising a C-terminal tail containing no DK sequence. In an exemplary embodiment, the C-terminal tail comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the C-terminal tail further comprises a cysteine residue. In other embodiments, the C-terminal tail comprises the sequence of SEQ ID NO: 5.

In certain embodiments, fibronectin based scaffold proteins bind to targets that are not bound by the wild type ¹⁰ Fn3 domain. In certain embodiments, the fibronectin based scaffold protein does not bind to one or more of EGFR, human serum albumin or PCSK9. In certain embodiments, a fibronectin based scaffold protein comprising a single ¹⁰ Fn3 domain does not bind to one or more of EGFR, human serum albumin or PCSK9. In certain embodiments, the multivalent fibronectin based scaffold protein comprises the following combinations of target molecules: i) EGFR and IGF-IR; ii) EGFR and any other target protein; or iii) human serum albumin and It does not bind to one or more of any other target proteins.

In some embodiments, the ¹⁰ Fn3 domain of a fibronectin based scaffold protein further comprises an N-terminal extension comprising 1-10 amino acids. In other embodiments, the ¹⁰ Fn3 domain of a fibronectin based scaffold protein comprises a sequence selected from the group consisting of: M, MG, G and any of SEQ ID NOs: 19-21 and 26-31.

In some embodiments, the scaffold proteins based on fibronectin, further comprises a second ^{10 Fn3} domain, here, said second ^{10 Fn3} domain, SEQ ID NO: having one at least 60% identity It comprises an amino acid sequence are those that bind to a target molecule with a K _D of less than 100 nM, and, said second ^{10 Fn3} domain are those further comprising a C-terminal tail containing no DK sequence. In an exemplary embodiment, the second ¹⁰ Fn3 domain comprises a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first and second ¹⁰ Fn3 domains bind to different targets. In some embodiments, the second ¹⁰ Fn3 domain further comprises an N-terminal extension comprising 1-10 amino acids. In some embodiments, the N-terminal extension comprises a sequence selected from the group consisting of: M, MG, G and any of SEQ ID NOs: 19-21 and 25-31. In some embodiments, the first and second ¹⁰ Fn3 domains are linked by a polypeptide linker comprising 1-30 amino acids. In some embodiments, the polypeptide linker is selected from the group consisting of a glycine-serine based linker, a glycine-proline based linker, a proline-alanine linker and an Fn based linker.

In certain embodiments, the fibronectin based scaffold protein comprises one or more ¹⁰ Fn3 domains, including loop, AB; loop, BC; loop, CD; loop, DE; loop, EF; and loop, FG. Each independently has at least one loop selected from BC, DE and FG with an altered amino acid sequence compared to the sequence of the corresponding loop of the human ¹⁰ Fn3 domain. In certain embodiments, the ¹⁰ Fn3 domain comprises an amino acid sequence that is at least 50, 60, 70, or 80% identical to a naturally occurring human ¹⁰ Fn3 domain represented by SEQ ID NO: 1. In an exemplary embodiment, the fibronectin based scaffold protein is a dimer comprising two ¹⁰ Fn3 domains.

  In another aspect, the application is based on a novel fibronectin based on reduced protein fragmentation compared to the fibronectin based scaffold protein dimer described in PCT patent application WO 2009/142773. A scaffold protein dimer is provided. In some embodiments, the fibronectin based scaffold protein dimer comprises the amino acid sequence of SEQ ID NO: 48.

In another aspect, the application provides a fibronectin based scaffold protein dimer having an N1-D1-C1-L-N2-D2-C2 structure. In certain embodiments, N1 and N2 are any N-terminal extension independently comprising 0-10 amino acids; D1 and D2 are (i) at least 95% identical to the amino acid sequence shown in SEQ ID NO: 2 a tenth fibronectin type III domain having ⁽¹⁰ Fn3), wherein the said ¹⁰ Fn3 domains are binding to IGF-IR with a _{K D} of less than 500 nM, and (ii) SEQ ID NO: shown in 3 a ¹⁰ Fn3 domain having an amino acid sequence at least 95% identity to the, here, the ¹⁰ Fn3 domain is one that binds to VEGFR2 with a _{K D} of less than 500 nM, independently selected from the group consisting of; L is a polypeptide linker comprising 0-30 amino acid residues; C1 comprises the amino acid sequence shown in SEQ ID NO: 4 As well as C2 comprises the amino acid sequence shown in SEQ ID NO: 4, 5 or 6.

In some embodiments, the fibronectin based scaffold protein dimer has a N1-D1-C1-L-N2-D2-C2 structure, wherein D1 is the amino acid sequence shown in SEQ ID NO: 2. When comprise ¹⁰ Fn3 domain having at least 95% identity, D2 SEQ ID NO: one in which the amino acid sequence shown in 3 and containing ¹⁰ Fn3 domain having at least 95% identity. In another embodiment, the fibronectin based scaffold protein dimer has a N1-D1-C1-L-N2-D2-C2 structure, wherein D1 comprises the amino acid sequence shown in SEQ ID NO: 3 includes a ¹⁰ Fn3 domain having at least 95% identity, D2 SEQ ID NO: is intended to include ¹⁰ Fn3 domain with two amino acid sequence shown in at least 95% identity.

  In some embodiments, the fibronectin based scaffold protein dimer has a N1-D1-C1-L-N2-D2-C2 structure, wherein C2 comprises the amino acid sequence of SEQ ID NO: 6. It is a waste.

  In some embodiments, the fibronectin based scaffold protein dimer has a N1-D1-C1-L-N2-D2-C2 structure, wherein N1 is M, MG, G and SEQ ID NO: It comprises an amino acid sequence selected from the group consisting of any one of 19-21 and 26-31. In an exemplary embodiment, N1 comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the fibronectin based scaffold protein dimer has a N1-D1-C1-L-N2-D2-C2 structure, wherein N2 is M, MG, G and SEQ ID NO: It comprises an amino acid sequence selected from the group consisting of any one of 19-21 and 26-31. In an exemplary embodiment, N2 comprises the amino acid sequence of SEQ ID NO: 20.

  In some embodiments, the fibronectin based scaffold protein dimer has an N1-D1-C1-L-N2-D2-C2 structure, wherein L is a glycine-serine based linker. A polypeptide linker selected from the group consisting of a glycine-proline based linker, a proline-alanine linker, and an Fn based linker. In other embodiments, L comprises the amino acid sequence of SEQ ID NO: 7.

  In some embodiments, the fibronectin based scaffold protein is one or more selected from a polyoxyalkylene moiety, human serum albumin binding protein, sialic acid, human serum albumin, transferrin, IgG, IgG binding protein and Fc fragment. A pharmacokinetic (PK) portion of In some embodiments, the PK moiety is a polyoxyalkylene moiety and the polyoxyalkylene moiety is polyethylene glycol (PEG). In some embodiments, the PEG moiety is covalently linked to a fibronectin based scaffold protein via a Cys or Lys amino acid. In some embodiments, the PEG is between about 0.5 kDa and about 100 kDa.

  In one aspect, the application provides a pharmaceutically acceptable composition comprising a fibronectin based scaffold protein. In some embodiments, the composition is essentially free of pyrogens. In some embodiments, the composition is substantially free of microbial contamination, thereby making it suitable for in vivo administration. The composition can be formulated, for example, for IV, IP or subcutaneous administration. In some embodiments, the composition includes a physiologically acceptable carrier. In some embodiments, the pH of the composition is between 4.0-6.5. In some embodiments, the pH of the composition is between 4.0-5.5. In other embodiments, the pH of the composition is 5.5. In other embodiments, the pH of the composition is 4.0. In some embodiments, the concentration of the fibronectin based scaffold protein is 5 mg / ml in the composition.

  In another aspect, the application provides a pharmaceutical formulation comprising a fibronectin based scaffold protein, wherein the formulation comprises at least 5 mg / ml fibronectin based scaffold protein and a pH of 4.0. And suitable for intravenous administration. In some embodiments, the pharmaceutical formulation is stable at 25 ° C. for 4 weeks. In some embodiments, the pharmaceutical formulation has a fragmentation of less than 4%. In some embodiments, the formulation has an aggregation of less than 4%.

  In another aspect, the application treats a hyperproliferative disorder in a subject comprising administering to the subject in need a therapeutically effective amount of any of the compositions described herein. Provide a way to do that.

  In another aspect, the application provides a nucleic acid encoding a fibronectin based scaffold protein as described herein. Also included are vectors containing polynucleotides for such proteins. Suitable vectors include, for example, expression vectors. A further aspect of the application provides a cell comprising a polynucleotide, vector or expression vector encoding a fibronectin based scaffold protein. The sequence is preferably optimized to maximize expression in the cell type used. In some embodiments, the expression is in microbial cells. In other embodiments, the expression is in mammalian cells. Preferably the expression is in E. coli. In one aspect, the cell expresses a fibronectin based scaffold protein. In one aspect, the polynucleotide encoding a fibronectin based scaffold protein is a codon optimized for expression in a selected cell type. Culturing host cells containing a nucleic, vector or expression vector comprising a nucleic acid encoding a fibronectin based scaffold protein, and a fibronectin based scaffold protein expressed from the culture. Also provided is a method of producing a fibronectin based scaffold protein as described herein comprising recovering.

Size exclusion-high performance liquid chromatography (SE-HPLC) data showing the amount of protein aggregation over time for two concentrations of PEGylated V / I (DK +) (SEQ ID NO: 22). PEGylated protein was stored at 4 ° C. for 12 months in 10 mM succinic acid, 5% sorbitol, pH 5.5 at a protein concentration of 3 mg / mL. SE-HPLC data showing the amount of protein fragmentation over time for two concentrations of PEGylated V / I (DK +) (SEQ ID NO: 22). PEGylated protein was stored at 4 ° C. for 12 months in 10 mM succinic acid, 5% sorbitol, pH 5.5 at a protein concentration of 3 mg / mL. SE-HPLC data showing the effect of pH on protein aggregation over time for PEGylated V / I (DK +) (SEQ ID NO: 22). PEGylated proteins were stored for 3 weeks at 25 ° C. in formulations containing 20 mM sodium acetate (for pH 4 and 5) or 20 mM sodium phosphate (for pH 6 and 7) and 50 mM NaCl. SE-HPLC data showing the effect of pH on protein fragmentation over time for PEGylated V / I (DK +) (SEQ ID NO: 22). PEGylated proteins were stored for 3 weeks at 25 ° C. in formulations containing 20 mM sodium acetate (for pH 4 and 5) or 20 mM sodium phosphate (for pH 6 and 7) and 50 mM NaCl. Reversed phase-high performance liquid showing the cleavage site of the PEGylated V / I (DK +) molecule (SEQ ID NO: 22) after storage at 25 ° C. for 4 weeks in 10 mM acetate, 150 mM NaCl at pH 5.5. Chromatographic (RP-HPLC) profile. As shown in the figure, protein cleavage occurs directly at the following positions, D95, D106, D180 and D200, and most occurs directly at the following positions, D95 and D200. The PEGylated protein was stored for 4 weeks at 25 ° C. in 10 mM sodium acetate, 150 mM sodium chloride, pH 5.5 at a protein concentration of 5 mg / mL. SE-HPLC data showing the effect of pH on protein aggregation over time for PEGylated E / I (DK +) (SEQ ID NO: 23). PEGylated protein was stored in 10 mM succinic acid, 5% sorbitol, pH 4.0, 4.5 and 5.5 for 4 weeks at 25 ° C. SE-HPLC data showing the effect of pH on the amount of protein fragmentation over time for PEGylated E / I (DK +) (SEQ ID NO: 23). PEGylated protein was stored in 10 mM succinic acid, 5% sorbitol, pH 4.0, 4.5 and 5.5 for 4 weeks at 25 ° C. The cleavage site of PEGylated E / I (DK +) (SEQ ID NO: 23) after storage at 25 ° C. for 4 weeks in 10 mM succinic acid, 5% sorbitol, pH 4.0 at a protein concentration of 5 mg / mL. Shown, RP-HPLC profile. As shown in the figure, protein cleavage occurs directly at the following positions, D95, D199 and D218. SE-HPLC data showing the effect of pH on protein aggregation over time for various fibronectin based scaffold protein constructs. Aggregation levels for various fibronectin based scaffold proteins were tested at either pH 5.5 or pH 4.0 during 4 weeks storage at 25 ° C. E / I (DK +) is SEQ ID NO: 23; E / I (DK−, without C-terminus) is SEQ ID NO: 24; E / I (2DK−) is SEQ ID NO: 25; V / I (DK +) is SEQ ID NO: 22. RP-HPLC data showing the amount of fragmentation of various fibronectin based scaffold proteins at various pHs during storage for 4 weeks at 25 ° C. Fibronectin based scaffold proteins containing DK sequences are more susceptible to fragmentation at pH 4.0 compared to pH 5.5. Fibronectin based scaffold proteins that do not contain DK sequences are more resistant to fragmentation at pH 4.0 compared to fibronectin based scaffold proteins that contain DK sequences. E / I (DK +) is SEQ ID NO: 23; E / I (DK−, without C-terminus) is SEQ ID NO: 24; E / I (2DK−) is SEQ ID NO: 25; V / I (DK +) is SEQ ID NO: 22. The major cleavage site in the E / I (DK−, no C-terminal) molecule (SEQ ID NO: 24) is D199, while the major cleavage site in the E / I (DK +) molecule (SEQ ID NO: 23) is D218. LC-MS data demonstrating that there is. LC-MS data demonstrating that the major cleavage site in the E / I (2DK-) molecule (SEQ ID NO: 25) is D199. Aggregation rate observed in VI (DK +) (SEQ ID NO: 56) and VI (DK−) (SEQ ID NO: 57) under storage at 25 ° C. for up to 2 months as assessed by SE-HPLC analysis. Clip rate observed in VI (DK +) (SEQ ID NO: 56) and VI (DK−) (SEQ ID NO: 57) under storage at 25 ° C. for up to 2 months as assessed by RP-HPLC analysis. Clip region of the overlaid chromatogram of RP-HPLC of VI (DK +) (SEQ ID NO: 56) and VI (DK-) (SEQ ID NO: 57) after 2 months incubation at 25 ° C. The total% clip for VI (DK +) is 16%, whereas for VI (DK−) it is 6.9%.

Detailed Description Definition By “polypeptide” is meant any sequence of two or more amino acids, regardless of length, post-translational modification or function. “Polypeptide”, “peptide” and “protein” are used interchangeably herein. Polypeptides can include natural and unnatural amino acids such as those described in US Pat. No. 6,559,126, which is incorporated herein by reference. Polypeptides may also be modified by any of a variety of standard chemical methods (eg, amino acids may be modified with protecting groups; the carboxy terminal amino acid may be terminal amide group). The amino terminal residue may be modified, for example, with a group to enhance lipophilicity; or the polypeptide may be chemically glycosylated, or may be stable or in vivo. May be modified to increase half-life). Polypeptide modifications include attachment of another structure to the polypeptide, such as a cyclic compound or other molecule, and one or more in an altered configuration (ie, R or S; or L or D) Polypeptides containing the amino acids of

  As used herein, “percent amino acid sequence identity (%)” refers to aligning sequences to achieve maximum percent sequence identity without considering any conservative substitutions as part of sequence identity, Defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the selected sequence, optionally after introducing gaps. For purposes of determining percent amino acid sequence identity, alignment can be performed in a variety of ways within the skill in the art, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. This can be accomplished using publicly available computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is available from Genentech, Inc. And by the United States Copyright Office, Washington D. C. , 20559, which is registered under US copyright registration number TXU510087, and is registered with Genentech, Inc. , South San Francisco, Calif. Publicly available through The ALIGN-2 program should be created for use with a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

  For purposes herein, a given amino acid sequence B and with respect thereto, or% amino acid sequence identity of a given amino acid sequence A (or with a given amino acid sequence B and with respect thereto, or (Which may be expressed as a given amino acid sequence A, which has or contains a specific% amino acid sequence identity) is calculated as follows: 100 × X / Y ratio, where X Is the number of amino acid residues scored as an identical match by the sequence alignment program ALIGN-2 in the alignment of the A and B programs, and Y is the total number of amino acid residues in B. Of course, if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. .

  The term “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the drug can reduce the number of cancer cells; it can reduce the size of the tumor; it can inhibit cancer cell invasion into peripheral organs (ie, it can be delayed to some extent, Preferably may be stopped); may inhibit tumor metastasis (ie may be delayed to some extent, preferably may be stopped); may be inhibited to some extent tumor growth; and / or may be associated with a disorder Symptoms can be reduced to some extent. So long as the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, in vivo efficacy can be measured, for example, by assessing time to disease progression (TTP) and / or determining response rate (RR).

  The half-life of an amino acid sequence or compound generally reduces the serum concentration of the polypeptide in vivo by 50%, for example, by degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. It can be defined as the time it takes. The half-life can be determined by any method known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art, for example, administering a suitable dose of an amino acid sequence or compound to a primate to be treated as appropriate; a blood sample or other sample from the primate on a regular basis Determining the level or concentration of the amino acid sequence or compound of the invention in the blood sample; and the data thus obtained (a plot of) from the level of the amino acid sequence or compound of the invention or It may include calculating the time until the concentration is reduced by 50% compared to the initial level at the time of administration. For example, Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmaceuticals and in Peters et al, Pharmacokine analysis: A PracticalAp. Marcel Dekker, 2nd Rev. Reference is also made to “Pharmacocetics” published by edition (1982), M Gibaldi & D Perron.

  Half-life can be expressed using parameters such as t1 / 2-α, t1 / 2-β and area under the curve (AUC). As used herein, “increased half-life” refers to any increase in any one of these parameters, eg, any two of these parameters, or essentially three of these parameters. Point to. “Increasing half-life” refers specifically to an increase in t1 / 2-β with or without an increase in t1 / 2-α and / or AUC or both.

Overview Described herein are improved fibronectin based scaffold proteins that are associated with increased stability. The fibronectin based scaffold proteins described herein include one or more tenth fibronectin type III domains that are modified to bind to one or more desired targets. The specification also includes two human 10th fibronectin type III domains, one modified to specifically bind to VEGFR2 and one modified to specifically bind to IGF-IR; An improved VEGFR2 / IGF-IR bispecific fibronectin based scaffold protein dimer has been described that is associated with increased stability. PCT patent application WO 2009/142773 describes fibronectin scaffold multimers that can be linked covalently or non-covalently and bind to both VEGFR2 and IGF-IR. . This application is a surprising discovery that, to some extent, bispecific fibronectin based scaffold proteins that bind to VEGFR2 and IGF-IR undergo high frequency fragmentation at specific aspartic acid residues. About. In particular, it has been discovered that aspartic acid residues directly followed by lysine residues are more sensitive to cleavage in fibronectin based scaffold proteins than aspartic acid residues followed by other amino acids. It was. The application also demonstrates the extent of fragmentation of scaffold proteins based on VEGFR2 / IGF-IR binding fibronectin, despite the same storage conditions and a high percentage of sequence identity shared between these similar proteins. The surprising discovery that the relevant fibronectin based scaffold proteins, ie, the degree of fragmentation associated with bispecific EGFR / IGF-IR binding fibronectin based scaffold protein dimers About. The application also shows that fragmentation of the model fibronectin based scaffold protein is significantly reduced when the DK site is modified or modified to replace aspartic acid residues with different amino acids such as glutamic acid. To demonstrate. Also provided herein are improved compositions of fibronectin-based scaffold proteins that have increased stability during storage.

Fibronectin-based scaffold Fn3 refers to a type III domain derived from fibronectin. The Fn3 domain is small, monomeric, soluble and stable. It lacks disulfide bonds and is stable under reducing conditions. The overall structure of Fn3 is similar to immunoglobulin. The Fn3 domain is, in order from the N-terminus to the C-terminus, β or β-like chain, A; loop, AB; β or β-like chain, B; loop, BC; β or β-like chain, C; loop, CD; β-like chain, D; loop, DE; β or β-like chain, E; loop, EF; β or β-like chain, F; loop, FG; and β or β-like chain, G. The seven antiparallel β strands are grouped together as two β sheets that form a stable core, while creating two “faces” composed of loops that bind to β or β-like strands. Loops AB, CD and EF are located on one face and loops BC, DE and FG are located on the opposite face. Any or all loops AB, BC, CD, DE, EF and FG may participate in ligand binding. There are at least 15 different Fn3 modules, and the sequence homology between modules is low, but they all share high similarity in tertiary structure.

Adnectins ^trademark (Adnexus, a Bristol-Myers Squibb Company) is 10th fibronectin type III domain, i.e. a ligand binding scaffold proteins based on tenth module ⁽¹⁰ Fn3) of Fn3. The amino acid sequence of naturally occurring human ¹⁰ Fn3 is shown in SEQ ID NO: 37:

  In SEQ ID NO: 37, the AB loop corresponds to residues 15-16, the BC loop corresponds to residues 21-30, the CD loop corresponds to residues 39-45, and the DE loop corresponds to residues 51-56 , The EF loop corresponds to residues 60-66, and the FG loop corresponds to residues 76-87. For example, Xu et al. , Chemistry & Biology 2002 9: 933-942. The BC, DE, and FG loops are aligned along one face of the molecule, and the AB, CD, and EF loops are aligned along the opposite face of the molecule. In SEQ ID NO: 37, β chain A corresponds to residues 9-14, β chain B corresponds to residues 17-20, β chain C corresponds to residues 31-38, and β chain D remains. Corresponding to groups 46-50, β chain E corresponds to residues 57-59, β chain F corresponds to residues 67-75, and β chain G corresponds to residues 88-94. The chains are connected to each other through corresponding loops, for example, chains A and B are connected through loop AB in the order of chain A, loop AB, chain B, etc. The first 8 amino acids of SEQ ID NO: 37 (above italics) can be deleted while retaining the binding activity of the molecule. In SEQ ID NO: 37, the residues involved in the formation of the hydrophobic core (“core amino acid residues”) are the following amino acids of SEQ ID NO: 37: L8, V10, A13, L18, I20, W22, Y32, I34. , Y36, F48, V50, A57, I59, L62, Y68, I70, V72, A74, I88, I90 and Y92, wherein the core amino acid residue is followed by a single letter amino acid code followed by Represented by the position in SEQ ID NO: 37 where they are located. For example, Dickinson et al. , J .; Mol. Biol. 236: 1079-1092 (1994).

^{The 10} Fn3 domain is structurally and functionally similar to an antibody, particularly the variable region of an antibody. ^{While 10} Fn3 domains can be described as “antibody mimics” or “antibody-like proteins”, they exhibit numerous advantages over conventional antibodies. In particular, they exhibit better folding and thermostable properties compared to antibodies and lack disulfide bonds known to interfere with or inhibit correct folding under certain conditions.

The BC, DE and FG loops of the ¹⁰ Fn3 domain are similar to complementarity determining regions (CDRs) from immunoglobulins. Changes in the amino acid sequence in these loop regions change the binding properties of ¹⁰ Fn3. A ¹⁰ Fn3 domain with modifications in the AB, CD and EF loops can also be made to generate molecules that bind to the desired target. The protein sequence outside the loop resembles an immunoglobulin-derived framework region and plays a role in the structural conformation of ¹⁰ Fn3. Changes in the framework-like region of ¹⁰ Fn3 are tolerated as long as the structural conformation is not altered enough to disrupt ligand binding. Methods for generating ¹⁰ Fn3 ligand-specific binders are described in PCT International Publication Nos. 00/034787, 01/64942, and 02/032925, which disclose high affinity TNFα binders, high affinity VEGFR2 binders. PCT International Publication No. 2008/097497 discloses PCT International Publication No. 2008/066752 which discloses high affinity IGF-IR binder, and PCT International Publication No. which discloses high affinity multivalent VEGFR2 / IGF-IR binder 2009/142773. Additional references discussing ¹⁰ Fn3 binders and methods for selecting binders include PCT WO 98/056915, 02/081497 and 2008/031098, and US Patent Application Publication No. 2003. No. 0186385.

As mentioned above, the amino acid residues corresponding to residues 21-30, 51-56 and 76-87 of SEQ ID NO: 37 define the BC, DE and FG loops, respectively. However, not all residues in the loop region need to be modified to obtain a ¹⁰ Fn3 binding domain with strong affinity for the desired target, such as VEGFR2 or IGF-IR. Should be understood. For example, in some embodiments, only residues corresponding to amino acids 23-29 in the BC loop, amino acids 52-55 in the DE loop, and amino acids 77-86 in the FG loop are modified to produce a high affinity ¹⁰ Fn3 binder. (See, for example, the VEGFR2 binding core having SEQ ID NO: 3). Thus, in certain embodiments, the BC loop is defined by the amino acid sequence corresponding to residues 23-29 of SEQ ID NO: 37, and the DE loop is defined by the amino acid sequence corresponding to residues 52-55 of SEQ ID NO: 37. And the FG loop is defined by the amino acid sequence corresponding to residues 77-86 of SEQ ID NO: 37.

Furthermore, insertions and deletions in the loop region can also be made while generating high affinity ¹⁰ Fn3 binding domains. For example, the FG loop of the VEGFR2 binder having SEQ ID NO: 3 has an FG loop of the same length as the wild type ¹⁰ Fn3 domain, ie, 10 residues 77-86 of SEQ ID NO: 37 are of SEQ ID NO: 3 10 residues 69-78 were exchanged. In contrast, the FG loop of the IGF-IR binder having SEQ ID NO: 2 is shorter in length than the corresponding FG loop of the wild type ¹⁰ Fn3 domain, ie, 10 residues 77-86 of SEQ ID NO: 37. Were replaced at 6 residues 69-74 of SEQ ID NO: 2. Finally, the FG loop of the EGFR binder having SEQ ID NO: 39 is shorter in length than the corresponding FG loop of the wild type ¹⁰ Fn3 domain, ie, the 10 residues 77-86 of SEQ ID NO: 37 are sequenced. Exchanged at 15 residues 69-83 of number: 39.

Thus, in some embodiments, one or more loops selected from BC, DE, and FG may be extended or shortened in length compared to the corresponding loop in wild type human ¹⁰ Fn3. In some embodiments, the length of the loop can be extended by 2-25 amino acids. In some embodiments, the loop length can be shortened by 1-11 amino acids. In particular, the FG loop of ¹⁰ Fn3 is 12 residues long while the corresponding loop in the antibody heavy chain ranges from 4 to 28 residues. Therefore, to optimize antigen binding, the length of the ¹⁰ Fn3 FG loop is varied in length as well as sequence to cover the range of 4-28 residue CDR3, and is most likely in antigen binding Gains mobility and affinity. In some embodiments, one or more residues of the integrin binding motif “arginine-glycine-aspartic acid” (RGD) (amino acids 78-80 of SEQ ID NO: 37) can be exchanged to disrupt integrin binding. . In one embodiment, the RGD sequence is exchanged by a polar amino acid-neutral amino acid-acidic amino acid sequence (N-terminal to C-terminal direction). In another embodiment, the RGD sequence is exchanged with SGE.

The non-ligand binding sequence of ¹⁰ Fn3, a “ ¹⁰ Fn3 scaffold” can be altered, provided that the ¹⁰ Fn3 domain retains ligand binding function and / or structural stability. In some embodiments, one or more of Asp7, Glu9, and Asp23 are replaced by other amino acids, such as, for example, non-negatively charged amino acid residues (eg, Asn, Lys, etc.). These mutations have been reported to have the effect of promoting greater stability of mutant ¹⁰ Fn3 at neutral pH compared to the wild type form (see PCT WO 02/04523). Wanna) Various further modifications in beneficial or intermediate ¹⁰ Fn3 scaffolds have been disclosed. For example, Batori et al. , Protein Eng. 2002 15 (12): 1015-20; Koide et al. Biochemistry 2001 40 (34): 10326-33.

^{The 10} Fn3 scaffold can be modified by one or more conservative substitutions. ^{As many} as 5%, 10%, 20%, or even 30% or more of the amino acids in the ¹⁰ Fn3 scaffold can be altered by conservative substitutions without substantially changing the affinity of ¹⁰ Fn3 for the ligand. In certain embodiments, the scaffold is about 0-15, 0-10, 0-8, 0-6, 0-5, 0-4, 0-3, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 5-15 or 5-10 storage Amino acid substitutions may be included. In exemplary embodiments, the scaffold modification preferably has a binding affinity of ¹⁰ Fn3 binder to the ligand of 1/100, 50/25, 10/5, or Reduce to less than half. Such changes may change the immunogenicity of ¹⁰ Fn3 in vivo, and such changes would be desirable if immunogenicity is reduced. As used herein, a “conservative substituent” is a residue that is physically or functionally similar to a corresponding reference residue. That is, conservative substituents and their reference residues have similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, and the like. Preferred conservative substitutions are described in Dayhoff et al. , Atlas of Protein Sequence and Structure 5: 345-352 (1978 & Supp.). Examples of conservative substitutions are substitutions within the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; ) Asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine.

In one aspect, the application provides a fibronectin based scaffold protein, eg, a polypeptide having a C-terminal tail that includes at least one ¹⁰ Fn3 domain and lacks a DK sequence. In an exemplary embodiment, a fibronectin based scaffold protein comprises a ¹⁰ Fn3 domain with a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 4. Such fibronectin based scaffold proteins have improved stability compared to fibronectin based scaffolds containing one or more DK sequences.

In some embodiments, the fibronectin based scaffold protein comprises a human ¹⁰ Fn3 domain having the amino acid sequence of SEQ ID NO: 1 and at least 40%, 50%, 55%, 60%, 65%, 70%, Contains ¹⁰ Fn3 domains with 75%, 80%, 85% or 90% identity. Most of the variability generally occurs in one or more loops. Each β or β-like chain of the ¹⁰ Fn3 domain in a fibronectin based scaffold protein has a corresponding β of SEQ ID NO: 1, provided that such variation does not disturb the stability of the polypeptide in physiological conditions. Or comprises, consists essentially of or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the sequence of the β-like chain. In the exemplary ^embodiment, 10 Fn3 domain, 500nM, 100nM, 1nM, 500pM , bind the desired target with 100pM or less than less _{K D.} In an exemplary embodiment, a fibronectin based scaffold protein specifically binds to a target that is not bound by a wild type ¹⁰ Fn3 domain, particularly a wild type human ¹⁰ Fn3 domain.

In some embodiments, the present disclosure ^provides a polypeptide comprising a 10 Fn3 domain, here, the ¹⁰ Fn3 domain, loop, AB; loop, BC; loop, CD; loop, DE; loop, EF; and having at least one loop selected from loop BC, DE and FG comprising a loop, FG, and comprising an amino acid sequence that is altered compared to the sequence of the corresponding loop of the human ¹⁰ Fn3 domain . In some embodiments, the BC and FG loops are changed. In some embodiments, the BC, DE and FG loops are altered, ie the ¹⁰ Fn3 domain comprises a non-naturally occurring loop. By “altered” is meant one or more amino acid sequence changes relative to the template sequence (ie, the corresponding human fibronectin domain), including amino acid additions, deletions and substitutions. Altering the amino acid sequence may generally be accomplished through intentional, blind or spontaneous sequence variation of the nucleic acid coding sequence and occurs by any technique, such as PCR, error-prone PCR, or chemical DNA synthesis. May be.

In some embodiments, one or more loops selected from BC, DE and FG may extend or shorten in length compared to the corresponding human fibronectin loop. In some embodiments, the loop length can be extended by 2-25 amino acids. In some embodiments, the loop length can be shortened by 1-11 amino acids. In particular, the FG loop of human ¹⁰ Fn3 is 12 residues long, while the corresponding loop in the antibody heavy chain ranges from 4-28 residues. Therefore, to optimize antigen binding, the length of the ¹⁰ Fn3 FG loop is varied in length as well as sequence to cover the range of 4-28 residue CDR3, and is most likely in antigen binding Gains mobility and affinity.

In some embodiments, the fibronectin based scaffold protein is a non-loop region of SEQ ID NO: 1 and at least 80, 85, 90, 95, wherein at least one selected BC, DE and FG are altered. It contains ¹⁰ Fn3 domains with 98 or 100% identical amino acid sequence. In some embodiments, the altered BC loop has up to 10 amino acid substitutions, up to 4 amino acid deletions, up to 10 amino acid insertions, or combinations thereof. In some embodiments, the altered DE loop has up to 6 amino acid substitutions, up to 4 amino acid deletions, up to 13 amino acid insertions, or combinations thereof. In some embodiments, the FG loop has up to 12 amino acid substitutions, up to 11 amino acid deletions, up to 25 amino acid insertions, or a combination thereof.

配列番号：３８において、ＢＣループはＸ_ｘにより表され、ＤＥループはＸ_ｙにより表され、かつＦＧループはＸ_ｚにより表される。Ｘは任意のアミノ酸を表し、Ｘに続く下付き文字はアミノ酸の数の整数値を表す。特に、ｘ、ｙおよびｚは、それぞれ独立して、約２−２０、２−１５、２−１０、２−８、５−２０、５−１５、５−１０、５−８、６−２０、６−１５、６−１０、６−８、２−７、５−７、または６−７個のアミノ酸の範囲であり得る。好ましい実施形態では、ｘは７アミノ酸、ｙは４アミノ酸、かつｚは６、１０または１５アミノ酸である。β鎖の配列（下線）は、配列番号：３８に示される対応するアミノ酸に比べて、７種の足場領域の全てにわたって、０から１０、０から８、０から６、０から５、０から４、０から３、０から２、または０から１の範囲で置換、欠失または付加を有し得る。例示的な実施形態では、β鎖の配列は、配列番号：３８に示される対応するアミノ酸に比べて、全ての７種の足場領域にわたって、０から１０、０から８、０から６、０から５、０から４、０から３、０から２、または０から１の範囲で保存的置換を有し得る。特定の実施形態では、コアアミノ酸残基は固定され、任意の置換、保存的置換、欠失または付加は、コアアミノ酸残基以外の残基で生じる。太字で示されるＥＩＥＫテール（配列番号：４）は固定される。特定の実施形態では、ループ領域を直接挟むアミノ酸（例えば、下線を引かれていない残基）は、それぞれ独立して、置換または欠失され得る。ループを直接挟む残基を置換する時、各残基は同数のアミノ酸を有する配列で、またはより多くのアミノ酸配列（例えば、０−１０、０−８、０−５、０−３もしくは０−２個のアミノ酸残基の挿入）で置換され得る。下線を引かれていない残基はループ領域の一部分であり、そのため、^１０Ｆｎ３ドメインの構造に著しく影響を与えることなく、置換を受け入れる。 In certain embodiments, fibronectin based scaffold proteins generally comprise a ¹⁰ Fn3 domain defined by the following sequence:

SEQ ID NO: In 38, BC loop is represented by _{X x,} DE loop is represented by _{X y,} and FG loop is represented by _{X z.} X represents any amino acid, and the subscript following X represents an integer value of the number of amino acids. In particular, x, y and z are each independently about 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20. 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. In preferred embodiments, x is 7 amino acids, y is 4 amino acids, and z is 6, 10 or 15 amino acids. The sequence of the β chain (underlined) is 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 0 over all seven scaffold regions compared to the corresponding amino acid shown in SEQ ID NO: 38. There may be substitutions, deletions or additions in the range of 4, 0 to 3, 0 to 2, or 0 to 1. In an exemplary embodiment, the sequence of the β chain is from 0 to 10, 0 to 8, 0 to 6, 0 to all 7 scaffold regions compared to the corresponding amino acid set forth in SEQ ID NO: 38. It may have conservative substitutions in the range of 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, the core amino acid residues are fixed and any substitutions, conservative substitutions, deletions or additions occur at residues other than the core amino acid residues. The EIEK tail (SEQ ID NO: 4) shown in bold is fixed. In certain embodiments, amino acids that directly span the loop region (eg, residues that are not underlined) can each be independently substituted or deleted. When substituting residues directly across the loop, each residue is a sequence having the same number of amino acids, or more amino acid sequences (eg, 0-10, 0-8, 0-5, 0-3 or 0- Substitution of two amino acid residues). Residues that are not underlined are part of the loop region and therefore accept substitutions without significantly affecting the structure of the ¹⁰ Fn3 domain.

^{The 10} Fn3 domain generally begins at amino acid number 1 of SEQ ID NO: 37. However, domains with amino acid deletions are also encompassed by the present invention. In some embodiments, the first 8 amino acids of SEQ ID NO: 37 are deleted. Additional sequences can also be added to the N or C terminus of the ¹⁰ Fn3 domain having amino acids corresponding to 1-94 of SEQ ID NO: 37 or amino acids 9-94 of SEQ ID NO: 37. For example, an additional MG sequence may be placed at the N-terminus of the ¹⁰ Fn3 domain. This M will normally be cleaved, leaving G at the N-terminus. In some embodiments, the N-terminal extension consists of an amino acid sequence selected from the group consisting of M, MG, G, and any of SEQ ID NOs: 19-21.

^{The 10} Fn3 domain optionally includes an N-terminal extension of 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2 or 1 amino acid in length obtain. Exemplary N-terminal extensions are M, MG, G, MGVSDVPRDL (SEQ ID NO: 19), VSDVPRDL (SEQ ID NO: 20) and GVSDVPRDL (SEQ ID NO: 21), or SEQ ID NO: N-terminal truncation of any one of 19, 20, or 21 is included. Other suitable N-terminal extensions are, for example, X _n SDVPRDL (SEQ ID NO: 26), X _n DVPRDL (SEQ ID NO: 27), X _n VPRDL (SEQ ID NO: 28), X _n PRDL (SEQ ID NO: 29) X _n RDL (SEQ ID NO: 30), X _n DL (SEQ ID NO: 31) or X _n L, where n = 0, 1 or 2 amino acids and n = 1, X is Met or Gly And when n = 2, X is Met-Gly. If a Met-Gly sequence is added to the N-terminus of the ¹⁰ Fn3 domain, this M will normally be cleaved, leaving a G at the N-terminus.

The fibronectin based scaffold protein provided herein comprises a ¹⁰ Fn3 domain with a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 4. Exemplary C-terminal tails include polypeptides that are 1-20, 1-15, 1-10, 1-8, 1-5 or 1-4 amino acids in length. Specific examples of tail sequences include, for example, EIEK (SEQ ID NO: 4), EGSGC (SEQ ID NO: 5), EIEKPCQ (SEQ ID NO: 6), EIEKPSQ (SEQ ID NO: 32), and EIEKP (SEQ ID NO: 33). A polypeptide comprising, consisting of, consisting of, consisting of, consisting of, EIEKPS (SEQ ID NO: 34) or EIEKPC (SEQ ID NO: 35). Such C-terminal sequences are referred to herein as tails or extensions and are further described herein. In an exemplary embodiment, the C-terminal tail comprises, consists or consists of the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, the C-terminal sequence lacks a DK sequence. In an exemplary embodiment, the C-terminal tail includes residues that facilitate modification with PEG, ie, lysine or cysteine residues. In a preferred embodiment, the C-terminal tail lacks the DK sequence and contains a cysteine residue.

In certain embodiments, a fibronectin based scaffold protein comprises a ¹⁰ Fn3 domain with both an N-terminal extension and a C-terminal tail. In some embodiments, a His6-tag may be placed at the N-terminus or C-terminus.

In an exemplary embodiment, a fibronectin based scaffold protein comprises a ¹⁰ Fn3 domain that binds to VEGFR2. In certain embodiments, the scaffold proteins based on fibronectin, bind to VEGFR2 with a _{K D} of less than 500 nM, BC loops SEQ ID NO: comprises the amino acid sequence of 43, DE SEQ ID NO: comprises the amino acid sequence of the 44 , The FG loop comprises the amino acid sequence of SEQ ID NO: 45, and the protein comprises a C-terminal tail lacking the DK sequence. In an exemplary embodiment, the C-terminal tail is EIEK (SEQ ID NO: 4), EGSGC (SEQ ID NO: 5), EIEKPCQ (SEQ ID NO: 6), EIEKPSQ (SEQ ID NO: 32), EIEKP (SEQ ID NO: 33). , EIEKPS (SEQ ID NO: 34) or EIEKPC (SEQ ID NO: 35). In a preferred embodiment, the C-terminal tail comprises EIEK (SEQ ID NO: 4) or EIEKPCQ (SEQ ID NO: 6).

In certain embodiments, the fibronectin based scaffold protein is a multivalent protein comprising two or more ¹⁰ Fn3 domains. For example, a multivalent fibronectin based scaffold protein may comprise 2, 3 or more ¹⁰ Fn3 domains covalently linked. In an exemplary embodiment, the fibronectin based scaffold protein is a divalent or dimeric protein comprising two ¹⁰ Fn3 domains. In certain embodiments, a multivalent fibronectin based scaffold protein comprises a first ¹⁰ Fn3 domain that binds to a first target molecule and a second ¹⁰ Fn3 domain that binds to a second target molecule. The first and second target molecules may be the same or different target molecules. If the first and second target molecules are the same, the ¹⁰ Fn3 domains, ie the binding loops, may be the same or different. Therefore, the first and second ¹⁰ Fn3 domains bind at the same target but at different epitopes.

In the exemplary embodiment, each ¹⁰ Fn3 domain scaffold proteins that multivalent fibronectin based binds 500 nM, 100 nM, 1 nM, 500 pM, the desired target with 100pM or less than less _{K D.} In an exemplary embodiment, each 10Fn3 domain of a multivalent fibronectin based scaffold protein specifically binds to a target that is not bound by a wild type ¹⁰ Fn3 domain, particularly a wild type human ¹⁰ Fn3 domain.

The ¹⁰ Fn3 domains in a multivalent fibronectin based scaffold protein can be joined by a polypeptide linker. Exemplary polypeptide linkers include polypeptides having 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3 or 1-2 amino acids. Specific examples of suitable polypeptide linkers are further described herein. In certain embodiments, the linker is a C-terminal tail polypeptide as described herein, an N-terminal extension polypeptide as described herein, a linker polypeptide as described below, or any of them It may be a combination.

In the case of multivalent fibronectin based scaffold proteins, preferably none of the ¹⁰ Fn3 domains contain a C-terminal tail containing a DK sequence. In an exemplary embodiment, a multivalent fibronectin based scaffold protein comprises two or more ¹⁰ Fn3 domains, wherein each domain comprises a C-terminal tail that does not contain a DK sequence. In certain embodiments, the multivalent fibronectin based scaffold protein comprises two or more ¹⁰ Fn3 domains, wherein each domain does not contain a DK sequence and is a PEG moiety, such as a lysine or cysteine residue. It includes a C-terminal tail containing residues suitable for addition. In certain embodiments, a multivalent fibronectin based scaffold protein comprises two or more ¹⁰ Fn3 domains, wherein each domain comprises a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the multivalent fibronectin based scaffold protein comprises two or more ¹⁰ Fn3 domains, wherein the N-terminal ¹⁰ Fn3 domain comprises a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 4; The terminal ¹⁰ Fn3 domains are EIEK (SEQ ID NO: 4), EGSGC (SEQ ID NO: 5), EIEKPCQ (SEQ ID NO: 6), EIEKPSQ (SEQ ID NO: 32), EIEKP (SEQ ID NO: 33), EIEKPS (SEQ ID NO: 34) or a C-terminal tail comprising the amino acid sequence of EIEKPC (SEQ ID NO: 35).

By altering the loop sequence of the ¹⁰ Fn3 domain, a fibronectin based scaffold protein can be generated that binds to any desired target. In exemplary embodiments, fibronectin based scaffold proteins provided herein that have increased stability bind to therapeutically desirable targets such as, for example, TNFα, VEGFR2, IGF-IR or EGFR. obtain. In certain embodiments, the fibronectin based scaffold protein does not bind to one or more of the following targets: EGFR, IGF-IR, HSA and PCSK9, or any of the aforementioned fragments. In certain embodiments, a fibronectin based scaffold protein comprising a single ¹⁰ Fn3 domain binds to one or more of the following targets: EGFR, IGF-IR, HSA and PCSK9, or any of the aforementioned fragments. do not do. In certain embodiments, a fibronectin based scaffold protein comprising a single ¹⁰ Fn3 domain binds to any of the following targets: EGFR, IGF-IR, HSA and PCSK9, or any of the aforementioned fragments. do not do. In certain embodiments, a fibronectin based scaffold protein comprising two ¹⁰ Fn3 domains does not bind to one or more of the following targets: EGFR, IGF-IR, HSA and PCSK9, or any of the aforementioned fragments . In certain embodiments, a fibronectin based scaffold protein comprising two ¹⁰ Fn3 domains is a combination of the following target molecules: i) EGFR and IGF-IR; ii) EGFR and any other target protein; or iii ) Does not bind to one or more of human serum albumin and any other target protein. In certain embodiments, a fibronectin based scaffold protein comprising two ¹⁰ Fn3 domains is a combination of the following target molecules: i) EGFR and IGF-IR; ii) EGFR and any other target protein; or iii ) Does not bind to any of human serum albumin and any other target protein.

Fibronectin based scaffold protein dimers In certain embodiments, the fibronectin based scaffold proteins described herein are dimers comprising two ¹⁰ Fn3 domains. In one embodiment, the application provides (i) a BC loop having the amino acid sequence of SEQ ID NO: 40, a DE loop having the amino acid sequence of SEQ ID NO: 41, an FG loop having the amino acid sequence of SEQ ID NO: 42, and a sequence A ¹⁰ Fn3 domain comprising a C-terminal tail comprising any one of the amino acid sequences of Nos .: 4-6 and binding to IGF-IR; and (ii) a BC loop having the amino acid sequence of SEQ ID NO: 43; A DE loop having the amino acid sequence of SEQ ID NO: 44, an FG loop having the amino acid sequence of SEQ ID NO: 45, and a C-terminal tail containing any one amino acid sequence of SEQ ID NOs: 4-6, and binds to VEGFR2. ^those, 10 Fn3 domains: including first and second ¹⁰ Fn3 domain is selected from the group consisting of, / A I fibronectin provide anchorage protein dimer based. In the exemplary embodiment, each of the VEGFR2 and IGF-IR binding ¹⁰ Fn3 domain binds 500 nM, 100 nM, 1 nM, 500 pM, its target with 100pM or less than less _{K D.}

In certain embodiments, a V / I fibronectin based scaffold protein dimer comprises an IGF-IR binding domain and a VEGFR2 binding domain in order from the N-terminus to the C-terminus. In an exemplary embodiment, a fibronectin based scaffold protein dimer comprises (i) a BC loop having the amino acid sequence of SEQ ID NO: 40, a DE loop having the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 42. A first ¹⁰ Fn3 domain comprising an FG loop having the amino acid sequence of: and a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 4 and binding to IGF-IR; and (ii) of SEQ ID NO: 43 A BC loop having an amino acid sequence, a DE loop having an amino acid sequence of SEQ ID NO: 44, an FG loop having an amino acid sequence of SEQ ID NO: 45, and a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 6 and binding to VEGFR2 A second ¹⁰ Fn3 domain. In the exemplary embodiment, each of the VEGFR2 and IGF-IR binding ¹⁰ Fn3 domain binds 500 nM, 100 nM, 1 nM, 500 pM, its target with 100pM or less than less _{K D.}

In certain embodiments, a V / I fibronectin based scaffold protein dimer comprises a VEGFR2 binding domain and an IGF-IR binding domain in order from the N-terminus to the C-terminus. In an exemplary embodiment, a fibronectin based scaffold protein dimer comprises (i) a BC loop having the amino acid sequence of SEQ ID NO: 43, a DE loop having the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 45. A first ¹⁰ Fn3 domain comprising an FG loop having the amino acid sequence of: and a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 4 and binding to VEGFR2; and (ii) the amino acid sequence of SEQ ID NO: 40 A BC loop having the amino acid sequence of SEQ ID NO: 41, an FG loop having the amino acid sequence of SEQ ID NO: 42, and a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 6 and binding to IGF-IR A second ¹⁰ Fn3 domain. In the exemplary embodiment, each of the VEGFR2 and IGF-IR binding ¹⁰ Fn3 domain binds 500 nM, 100 nM, 1 nM, 500 pM, its target with 100pM or less than less _{K D.}

In certain embodiments, the V / I fibronectin based scaffold protein dimer has (i) at least 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2. Comprising, having, or consisting of an amino acid sequence having, and comprising, becoming, or consisting of a C-terminal tail having any one of the amino acid sequences of SEQ ID NOs: 4-6, in IGF-IR A ¹⁰ Fn3 domain that binds; and (ii) an amino acid sequence having at least 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 3, Or comprising, consisting of, or consisting of a C-terminal tail having any one amino acid sequence of SEQ ID NO: 4-6 , One that binds to ^VEGFR2, 10 Fn3 domains: including first and second ¹⁰ Fn3 domain is selected from the group consisting of. In the exemplary embodiment, each of the VEGFR2 and IGF-IR binding ¹⁰ Fn3 domain binds 500 nM, 100 nM, 1 nM, 500 pM, its target with 100pM or less than less _{K D.} In an exemplary embodiment, the first and second ¹⁰ Fn3 domains are linked via a polypeptide linker. In certain embodiments, a V / I fibronectin based scaffold protein dimer comprises an N-terminal VEGFR2 binding ¹⁰ Fn3 domain and a C-terminal IGF-IR binding ¹⁰ Fn3 domain. In certain embodiments, a V / I fibronectin based scaffold protein dimer comprises an N-terminal IGF-IR binding ¹⁰ Fn3 domain and a C-terminal VEGFR2 binding ¹⁰ Fn3 domain. In certain embodiments, the N-terminal ¹⁰ Fn3 domain comprises a C-terminal tail having the amino acid sequence of SEQ ID NO: 4, and the C-terminal ¹⁰ Fn3 domain comprises a C-terminal tail having the amino acid sequence of SEQ ID NO: 6.

  In certain embodiments, a V / I fibronectin based scaffold protein dimer comprises the amino acid sequence of any one of SEQ ID NOs: 48-55. In other embodiments, the V / I fibronectin based scaffold protein dimer comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the V / I fibronectin based scaffold protein dimer has an amino acid sequence of any one of SEQ ID NOs: 48-55 and at least 70, 75, 80, 85, 90, 95, Includes amino acid sequences with 97, 98, 99 or 100% identity.

In certain embodiments, a V / I fibronectin based scaffold protein dimer comprises a polypeptide having a N1-D1-C1-L-N2-D2-C2 structure, wherein N1 and N2 are 0. Any N-terminal extension independently comprising −10 amino acids, wherein D1 and D2 are (i) at least 90%, 95%, 97%, 98%, 99% with the amino acid sequence shown in SEQ ID NO: 2 or 10th fibronectin type III domain having 100% identity ⁽¹⁰ Fn3), wherein, the said ¹⁰ Fn3 domains are those that bind 500 nM, 100 nM, 1 nM, 500 pM, the IGF-IR at 100pM less or less a _{K D} And (ii) at least 90%, 95%, 97%, 98%, 99% with the amino acid sequence shown in SEQ ID NO: 3 Here ¹⁰ Fn3 domain, with 100% identity, the ¹⁰ Fn3 domain is one that binds to VEGFR2 with a _{K D} of less than 500 nM: it is independently selected from the group consisting of; L is 0-30 amino acid residues A polypeptide linker comprising a group; C1 comprises, comprises or consists of the amino acid sequence of SEQ ID NO: 4; C2 comprises any one of the amino acid sequences of SEQ ID NO: 4, 5 or 6; It consists of or consists of. In certain embodiments, D1 binds to VEGFR2, and D2 binds to IGF-IR. In other embodiments, D1 binds to IGF-IR and D2 binds to VEGFR2.

In certain embodiments, the D1 or D2 region comprises a BC loop having the amino acid sequence of SEQ ID NO: 43, a DE loop having the amino acid sequence of SEQ ID NO: 44, and an FG loop having the amino acid sequence of SEQ ID NO: 45. , a ¹⁰ Fn3 domain that binds to VEGFR2, here, the ¹⁰ Fn3 domain is one that binds to VEGFR2 with a _{K D} of less than 100 nM.

配列番号：３において、ＢＣ、ＤＥおよびＦＧループは太字で示される固定された配列を有し（例えば、配列番号：４３のアミノ酸配列を有するＢＣループ、配列番号：４４のアミノ酸配列を有するＤＥループ、および配列番号：４５のアミノ酸配列を有するＦＧループ）、かつ、下線が引かれた残りの配列（例えば、７つのβ鎖ならびにＡＢ、ＣＤおよびＥＦループの配列）は、配列番号：３に示される対応するアミノ酸と比べて、０から２０、０から１５、０から１０、０から８、０から６、０から５、０から４、０から３、０から２、または０から１の範囲で置換、保存的置換、欠失または付加を有する。特定の実施形態では、コアアミノ酸残基は固定され、任意の置換、保存的置換、欠失または付加は、コアアミノ酸残基以外の残基で生じる。 In certain embodiments, the D1 or D2 region is a VEGFR2 binder represented by the following amino acid sequence:

In SEQ ID NO: 3, the BC, DE and FG loops have a fixed sequence shown in bold (for example, the BC loop having the amino acid sequence of SEQ ID NO: 43, the DE loop having the amino acid sequence of SEQ ID NO: 44) And the FG loop having the amino acid sequence of SEQ ID NO: 45), and the remaining underlined sequences (eg, the sequences of the seven β chains and the AB, CD and EF loops) are shown in SEQ ID NO: 3. Range from 0 to 20, 0 to 15, 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1, compared to the corresponding amino acid With substitutions, conservative substitutions, deletions or additions. In certain embodiments, the core amino acid residues are fixed and any substitutions, conservative substitutions, deletions or additions occur at residues other than the core amino acid residues.

The ¹⁰ Fn3 domain that binds to VEGFR2 has an N-terminal extension of 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2 or 1 amino acid in length It can be optionally linked to (N1 or N2). Exemplary N-terminal extensions are M, MG, G, MGVSDVPRDL (SEQ ID NO: 19), VSDVPRDL (SEQ ID NO: 20) and GVSDVPRDL (SEQ ID NO: 21), or SEQ ID NO: N-terminal truncation of any one of 19, 20, or 21 is included. Other suitable N-terminal extensions are, for example, X _n SDVPRDL (SEQ ID NO: 26), X _n DVPRDL (SEQ ID NO: 27), X _n VPRDL (SEQ ID NO: 28), X _n PRDL (SEQ ID NO: 29) X _n RDL (SEQ ID NO: 30), X _n DL (SEQ ID NO: 31) or X _n L, where n = 0, 1 or 2 amino acids and n = 1, X is Met or Gly And when n = 2, X is Met-Gly. In a preferred embodiment, N1 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 19. In a preferred embodiment, N2 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 20.

The ¹⁰ Fn3 domain that binds to VEGFR2 can optionally include a C-terminal tail (C1 or C2). The C-terminal tail of the claimed protein fibronectin-based scaffold protein dimer does not contain a DK sequence. Exemplary C-terminal tails include polypeptides that are 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2 or 1 amino acid in length Can be mentioned. Specific examples of the C-terminal tail include EIEKPSQ (SEQ ID NO: 32), EIEKPCQ (SEQ ID NO: 6) and EIEK (SEQ ID NO: 4). In other embodiments, a suitable C-terminal tail is, for example, one of the following amino acid sequences (represented by the single letter amino acid code): EIE, EIEKP (SEQ ID NO: 33), EIEKPS (SEQ ID NO: 34) or EIEKPC It may be a C-terminal truncated fragment of SEQ ID NO: 4, 6 or 32, including (SEQ ID NO: 35). Other suitable C-terminal tails include, for example, ES, EC, EGS, EGC, EGSGS (SEQ ID NO: 36) or EGSGC (SEQ ID NO: 5). In certain embodiments, C1 comprises, consists of or consists of the amino acid sequence of SEQ ID NO: 4. In certain embodiments, C2 comprises, consists of or consists of the amino acid sequence of SEQ ID NO: 4, 5 or 6. In a preferred embodiment, C1 comprises, consists or consists of the amino acid sequence of SEQ ID NO: 4, and C2 comprises, consists of, consists of, or consists of the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the ¹⁰ Fn3 domain that binds to VEGFR2 comprises both an N-terminal extension and a C-terminal tail. In an exemplary embodiment, N1 begins with Gly or Met-Gly, C1 does not contain a cysteine residue, N2 does not start with Met, and C2 contains a cysteine residue. Specific examples of ¹⁰ Fn3 domains that bind to VEGFR2 are: (i) the amino acid sequence shown in SEQ ID NO: 3 or (ii) the amino acid sequence shown in SEQ ID NO: 3 and at least 85%, 90%, 95%, 97% , A polypeptide comprising an amino acid sequence having 98% or 99% identity.

In certain embodiments, the D1 or D2 region comprises a BC loop having the amino acid sequence of SEQ ID NO: 40, a DE loop having the amino acid sequence of SEQ ID NO: 41, and an FG loop having the amino acid sequence of SEQ ID NO: 42. , a ¹⁰ Fn3 domain binding to IGF-IR, wherein the, the ¹⁰ Fn3 domain are those binding to IGF-IR with a _{K D} of less than 100 nM.

配列番号：２において、ＢＣ、ＤＥおよびＦＧループは太字で示される固定された配列を有し（例えば、配列番号：４０のアミノ酸配列を有するＢＣループ、配列番号：４１のアミノ酸配列を有するＤＥループ、および配列番号：４２のアミノ酸配列を有するＦＧループ）、かつ、下線が引かれた残りの配列（例えば、７つのβ鎖ならびにＡＢ、ＣＤおよびＥＦループの配列）は、配列番号：２に示される対応するアミノ酸と比べて、０から２０、０から１５、０から１０、０から８、０から６、０から５、０から４、０から３、０から２、または０から１の範囲で置換、保存的置換、欠失または付加を有する。特定の実施形態では、コアアミノ酸残基は固定され、任意の置換、保存的置換、欠失または付加は、コアアミノ酸残基以外の残基で生じる。 In certain embodiments, the D1 or D2 region is an IGF-IR binder represented by the following amino acid sequence:

In SEQ ID NO: 2, the BC, DE and FG loops have a fixed sequence shown in bold (for example, the BC loop having the amino acid sequence of SEQ ID NO: 40, the DE loop having the amino acid sequence of SEQ ID NO: 41) And the FG loop having the amino acid sequence of SEQ ID NO: 42) and the remaining underlined sequences (eg, the sequences of the seven β chains and the AB, CD and EF loops) are shown in SEQ ID NO: 2. Range from 0 to 20, 0 to 15, 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1, compared to the corresponding amino acid With substitutions, conservative substitutions, deletions or additions. In certain embodiments, the core amino acid residues are fixed and any substitutions, conservative substitutions, deletions or additions occur at residues other than the core amino acid residues.

The ¹⁰ Fn3 domain that binds to IGF-IR has a length of 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acid in length. It can optionally be linked to a terminal extension (N1 or N2). Exemplary N-terminal extensions are M, MG, G, MGVSDVPRDL (SEQ ID NO: 19), VSDVPRDL (SEQ ID NO: 20) and GVSDVPRDL (SEQ ID NO: 21), or SEQ ID NO: N-terminal truncation of any one of 19, 20, or 21 is included. Other suitable N-terminal extensions are, for example, X _n SDVPRDL (SEQ ID NO: 26), X _n DVPRDL (SEQ ID NO: 27), X _n VPRDL (SEQ ID NO: 28), X _n PRDL (SEQ ID NO: 29) X _n RDL (SEQ ID NO: 30), X _n DL (SEQ ID NO: 31) or X _n L, where n = 0, 1 or 2 amino acids and n = 1, X is Met or Gly And when n = 2, X is Met-Gly. In a preferred embodiment, N1 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 19. In a preferred embodiment, N2 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 20.

The ¹⁰ Fn3 domain that binds to IGF-IR can optionally include a C-terminal tail (C1 or C2). The C-terminal tail of the claimed protein fibronectin-based scaffold protein dimer does not contain a DK sequence. Exemplary C-terminal tails include polypeptides that are 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2 or 1 amino acid in length Can be mentioned. Specific examples of the C-terminal tail include EIEKPSQ (SEQ ID NO: 32), EIEKPCQ (SEQ ID NO: 6) and EIEK (SEQ ID NO: 4). In other embodiments, a suitable C-terminal tail is, for example, one of the following amino acid sequences (represented by the single letter amino acid code): EIE, EIEKP (SEQ ID NO: 33), EIEKPS (SEQ ID NO: 34) or EIEKPC It may be a C-terminal truncated fragment of SEQ ID NO: 4, 6 or 32, including (SEQ ID NO: 35). Other suitable C-terminal tails include, for example, ES, EC, EGS, EGC, EGSGS (SEQ ID NO: 36) or EGSGC (SEQ ID NO: 5). In certain embodiments, C1 comprises, consists of or consists of the amino acid sequence of SEQ ID NO: 4. In certain embodiments, C2 comprises, consists of or consists of the amino acid sequence of SEQ ID NO: 4, 5 or 6. In a preferred embodiment, C1 comprises, consists or consists of the amino acid sequence of SEQ ID NO: 4, and C2 comprises, consists of, consists of, or consists of the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the ¹⁰ Fn3 domain that binds to IGF-IR comprises both an N-terminal extension and a C-terminal tail. In an exemplary embodiment, N1 begins with Gly or Met-Gly, C1 does not contain a cysteine residue, N2 does not start with Met, and C2 contains a cysteine residue. Specific examples of ¹⁰ Fn3 domains that bind to IGF-IR are: (i) the amino acid sequence shown in SEQ ID NO: 2 or (ii) the amino acid sequence shown in SEQ ID NO: 2 and at least 85%, 90%, 95%, A polypeptide comprising an amino acid sequence having 97%, 98% or 99% identity.

  The L region is a polypeptide linker. Exemplary polypeptide linkers include polypeptides having 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3 or 1-2 amino acids. Specific examples of suitable polypeptide linkers are further described herein. In certain embodiments, the linker may be a C-terminal tail polypeptide as described herein, an N-terminal extension polypeptide as described herein, or a combination thereof.

  In certain embodiments, one or more of N1, N2, L, C1 or C2 may comprise amino acid residues suitable for PEGylation, such as cysteine or lysine residues. In an exemplary embodiment, C2 comprises at least one amino acid suitable for PEGylation, such as a cysteine or lysine residue. Specific examples of suitable polypeptide linkers are further described below. Specific examples of scaffold protein dimers based on fibronectin having N1-D1-C1-L-N2-D2-C2 structure are: (i) Amino acid sequence shown in any one of SEQ ID NOs: 48-55 Or (ii) a polypeptide comprising an amino acid sequence having at least 85%, 90%, 95%, 97%, 98% or 99% identity with any one of SEQ ID NOs: 48-55.

配列番号：３８において、ＢＣループはＸ_Ｘにより表され、ＤＥループはＸ_ｙにより表され、かつＦＧループはＸ_ｚにより表される。β鎖の配列（下線）は、配列番号：３８に示される対応するアミン酸に比べて、７種の足場領域の全てにわたって、０から１０、０から８、０から６、０から５、０から４、０から３、０から２、または０から１の範囲で置換、保存的置換、欠失または付加を有し得る。例示的な実施形態では、β鎖の配列は、配列番号：３８に示される対応するアミン酸に比べて、全ての７種の足場領域にわたって、０から１０、０から８、０から６、０から５、０から４、０から３、０から２、または０から１の範囲で保存的置換を有し得る。特定の実施形態では、コアアミノ酸残基は固定され、任意の置換、保存的置換、欠失または付加は、コアアミノ酸残基以外の残基で生じる。太字で示されるＥＩＥＫ配列（配列番号：４）は固定される。特定の実施形態では、ループ領域を直接挟むアミノ酸（例えば、下線を引かれていない残基）は、それぞれ独立して、置換または欠失される。ループを直接挟む残基を置換する時、各残基は同数のアミノ酸を有する配列で、またはより多くのアミノ酸配列（例えば、０−１０、０−８、０−５、０−３もしくは０−２個のアミノ酸残基の挿入）で置換され得る。下線を引かれていない残基はループ領域の一部分であり、そのため、^１０Ｆｎ３ドメインの構造に著しく影響を与えることなく、置換を受け入れる。 In certain embodiments, a fibronectin based scaffold protein dimer has an N1-D1-L-N2-D2 structure, wherein D1 and D2 each comprise an amino acid sequence having the following sequence: It is a waste:

SEQ ID NO: In 38, BC loop is represented by _{X X,} DE loop is represented by _{X y,} and FG loop is represented by _{X z.} The sequence of the β chain (underlined) is 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 over all seven scaffold regions compared to the corresponding amino acid shown in SEQ ID NO: 38. Can have substitutions, conservative substitutions, deletions or additions in the range of 0 to 4, 0 to 3, 0 to 2, or 0 to 1. In an exemplary embodiment, the sequence of the β chain is 0 to 10, 0 to 8, 0 to 6, 0 over all seven scaffold regions compared to the corresponding amino acid shown in SEQ ID NO: 38. May have conservative substitutions in the range of from 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, the core amino acid residues are fixed and any substitutions, conservative substitutions, deletions or additions occur at residues other than the core amino acid residues. The EIEK sequence (SEQ ID NO: 4) shown in bold is fixed. In certain embodiments, amino acids that directly span the loop region (eg, residues that are not underlined) are each independently substituted or deleted. When substituting residues directly across the loop, each residue is a sequence having the same number of amino acids, or more amino acid sequences (eg, 0-10, 0-8, 0-5, 0-3 or 0- Substitution of two amino acid residues). Residues that are not underlined are part of the loop region and therefore accept substitutions without significantly affecting the structure of the ¹⁰ Fn3 domain.

In certain embodiments, a fibronectin based scaffold protein dimer has a N1-D1-L-N2-D2 structure, wherein D1 and D2 include: (i) ¹⁰ Fn3 comprising SEQ ID NO: 38 A domain, wherein X _X comprises, is essential, or consists of the amino acid sequence of SEQ ID NO: 40, X _y comprises, is essential, or consists of the amino acid sequence of SEQ ID NO: 41, and X _z Comprises, consists of or consists of the amino acid sequence of SEQ ID NO: 42, and (ii) a ¹⁰ Fn3 domain comprising SEQ ID NO: 38, wherein _XX is the amino acid sequence of SEQ ID NO: 43 wherein the result as an essential or consists, X _y SEQ ID NO: comprises 44 amino acid sequence of, become as essential or consist, and X _z is SEQ ID NO: 4 Comprising the amino acid sequence, comprising as essential, or is made of, are those selected from the group consisting of.

In an exemplary embodiment, a fibronectin based scaffold protein dimer has the N1-D1-L-N2-D2 structure, wherein D1 comprises the amino acid sequence of SEQ ID NO: 38, wherein _XX is Comprising, consisting of, or consisting of the amino acid sequence of SEQ ID NO: 40, X _y comprising, essential, or consisting of the amino acid sequence of SEQ ID NO: 41, and X _z being the amino acid sequence of SEQ ID NO: 42 the containing, be those made as essential, or consists, and, D2 is SEQ ID NO: 38 comprises the amino acid sequence of, here, X _X is SEQ ID NO: comprises 43 amino acid sequence of, become as essential, or consists, X _y SEQ ID NO: 44 comprises the amino acid sequence of, become as essential or consist, and X _z is SEQ ID NO: comprises the amino acid sequence of 45, it was essential , Or is made of.

In another embodiment, the fibronectin based scaffold protein dimer has an N1-D1-L-N2-D2 structure, wherein D1 comprises the amino acid sequence of SEQ ID NO: 38, wherein _XX is the sequence ID NO: 43 comprises the amino acid sequence of, become as essential or consists, X _y SEQ ID NO: comprises the amino acid sequence of 44, becomes as essential or consist, and X _z is SEQ ID NO: the amino acid sequence of the 45 including, made as essential, or made as in a from and, D2 is SEQ ID NO: 38 comprises the amino acid sequence of, here, X _X is SEQ ID NO: comprises the amino acid sequence of 40, becomes as essential or color, becomes, X _y SEQ ID NO: comprises 41 amino acid sequence of, become as essential or consists, is and X _z comprises SEQ ID NO: 42 amino acid sequence of comprised as essential, It is made of Taha.

  In certain embodiments, a fibronectin based scaffold protein dimer having an N1-D1-L-N2-D2 structure further comprises a C-terminal tail. In an exemplary embodiment, the C-terminal tail includes a residue suitable for addition of a PEG moiety, such as a lysine or cysteine residue. In a preferred embodiment, the C-terminal tail comprises the sequence PCQ.

  In various embodiments, the L region of a fibronectin based scaffold protein dimer having an N1-D1-L-N2-D2 structure is a polypeptide linker. Exemplary polypeptide linkers include polypeptides having 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3 or 1-2 amino acids. Specific examples of suitable polypeptide linkers are further described herein. In addition, N1 and N2 are the N-terminal extensions described herein above.

  In preferred embodiments, the fibronectin based scaffold protein dimers described herein have increased stability either in vitro, in vivo or both. In certain embodiments, the fibronectin based scaffold protein dimers described herein have reduced fragmentation and / or reduced aggregation during storage in solution. In certain embodiments, the fibronectin-based scaffold protein dimer described herein has an increased serum half-life.

In an exemplary embodiment, a fibronectin based scaffold protein dimer described herein exhibits reduced fragmentation compared to a fibronectin based scaffold protein dimer comprising a DK sequence. Have. In particular, the fibronectin-based scaffold protein dimer described herein comprises one or more DK sequences in any one of a C-terminal tail, an N-terminal extension, or a linker between two ¹⁰ Fn3 domains. Compared to a fibronectin based scaffold protein dimer with For example, a fibronectin based scaffold protein dimer includes, for example, a DK sequence in one or both C-terminal tail regions, including a tail having SEQ ID NO: 46 after the first and / or second ¹⁰ Fn3 subunits. It is more stable than a fibronectin based scaffold protein dimer with In an exemplary embodiment, the fibronectin based scaffold protein dimer described herein is a N1-D1-C1-L-N2-D2 wherein C1 and / or C2 comprises SEQ ID NO: 46. -Has reduced fragmentation compared to fibronectin based scaffold protein dimers with the C2 formula. Fragmentation can be assessed using, for example, the RP-HPLC analysis described in Example 3.

  In an exemplary embodiment, the fibronectin based scaffold protein dimer described herein is 7%, 6%, 5% during storage in solution at pH 4.0 for 4 weeks, Shows 4%, 3.5%, 3%, less than 2% or less fragmentation. In certain embodiments, the fibronectin based scaffold protein dimer described herein is an equivalent version of a fibronectin based scaffold protein dimer containing one or more DK sequences. Compared to, the level of fragmentation is reduced by at least 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or more.

  In an exemplary embodiment, a fibronectin-based scaffold protein dimer described herein is an equivalent serum half of a fibronectin-based scaffold protein dimer containing one or more DK sequences. The serum half-life is increased by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or more compared to the period. In other embodiments, the fibronectin based scaffold protein dimer described herein is an equivalent serum half-life of a fibronectin based scaffold protein dimer containing one or more DK sequences. 2, 3 times, 5 times, 10 times or more increased serum half-life.

In certain embodiments, the application provides an E / I fibronectin based scaffold protein dimer comprising one ¹⁰ Fn3 domain that binds to EGFR and one ¹⁰ Fn3 domain that binds to IGF-IR. . In certain embodiments, the E / I fibronectin based scaffold protein dimer comprises any one amino acid sequence of SEQ ID NOs: 22-25. In other embodiments, the E / I fibronectin based scaffold protein dimer comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the E / I fibronectin based scaffold protein dimer has an amino acid sequence of any one of SEQ ID NOs: 22-25 and at least 70, 75, 80, 85, 90, 95, 97. , 98, 99 or 100% identity.

Polypeptide Linkers The present application provides multivalent fibronectin based scaffold protein dimers comprising at least two ¹⁰ Fn3 domains linked via a polypeptide linker. In one embodiment, the application provides a multivalent fibronectin based scaffold protein dimer comprising at least two ¹⁰ Fn3 domains linked via a polypeptide linker (L). Polypeptide comprises a C-terminal domain containing a N-terminal domain and a second ¹⁰ Fn3 domain comprises a first ¹⁰ Fn3 domain. The first and second ¹⁰ Fn3 domains can be linked directly or indirectly through a polypeptide linker (L). An additional linker or spacer, eg, SEQ ID NO: 4, 6, or 32, may be introduced at the C-terminus of the first ¹⁰ Fn3 domain between the ¹⁰ Fn3 domain and the polypeptide linker. An additional linker or spacer may be introduced at the N-terminus of the second ¹⁰ Fn3 domain between the ¹⁰ Fn3 domain and the polypeptide linker.

^As a suitable linker for linking ¹⁰ Fn3 domains, it allows separate domains to fold independently of each other to form a three-dimensional structure that allows high affinity binding to the target molecule. There is something. The present application includes suitable linkers that meet these conditions include glycine-serine based linkers, glycine-proline based linkers, proline-alanine based linkers, and linker SEQ ID NO: 7. To provide that. The example described in WO2009 / 142773 demonstrates that Fn3 domains linked via these linkers retain their target binding function. In some embodiments, the linker is a glycine-serine based linker. These linkers contain glycine and serine residues and can be between 8-50, 10-30 and 10-20 amino acids long. Examples of such linkers include SEQ ID NOs: 8-12. In some embodiments, the polypeptide linker is selected from SEQ ID NOs: 8 and 9. In some embodiments, the linker is a glycine-proline based linker. These linkers contain glycine and proline residues and can be between 3-30, 10-30 and 3-20 amino acids long. Examples of such linkers include SEQ ID NOs: 13, 14, and 15. In some embodiments, the linker is a proline-alanine based linker. These linkers contain proline and alanine residues and can be between 3-30, 10-30, 3-20 and 6-18 amino acids in length. Examples of such linkers include SEQ ID NOs: 16, 17, and 18. It is believed that the optimal linker length and amino acid composition can be determined by routine experimentation by methods well known in the art. In some embodiments, the polypeptide linker is SEQ ID NO: 7. In an exemplary embodiment, the linker does not contain any DK sequences.

Pharmacokinetic moiety In one aspect, the application provides a fibronectin based scaffold protein further comprising a pharmacokinetic (PK) moiety. Pharmacokinetics encompasses, for example, the properties of a compound, including absorption, distribution, metabolism and excretion by a subject. Improved pharmacokinetics can be assessed according to recognized therapeutic needs. It is often desirable to increase bioavailability and / or increase the time between doses, possibly by increasing the time that the protein remains available in the serum after administration. In some cases, it is desirable to improve the continuity of the serum concentration of the protein over time (eg, reducing the difference in serum concentration of the protein immediately after administration and immediately before the next administration). Fibronectin-based scaffold proteins bind a moiety that reduces the clearance rate of the polypeptide in mammals (eg, mouse, rat or human) by more than three times compared to the unmodified polypeptide. Also good. Another measure of improved pharmacokinetics can include serum half-life, which is often divided into an alpha phase and a beta phase. Either or both phases can be greatly improved by the addition of appropriate moieties. A PK moiety refers to any protein, peptide or moiety that, when fused with a biologically active molecule, affects the pharmacokinetic properties of the biologically active molecule.

  PK moieties that tend to delay protein clearance from the blood include polyoxyalkylene moieties such as polyethylene glycol, sugars (eg sialic acid) and well tolerated protein moieties (eg Fc, Fc fragments, transferrin) Or serum albumin). A fibronectin based scaffold protein may be fused to albumin or a fragment (part) or variant of albumin as described in US Publication No. 20070048282. In some embodiments, the PK moiety is a serum albumin binding protein, such as those described in US Publication Nos. 2007/0178082 and 2007/0269422. In some embodiments, the PK moiety is a serum immunoglobulin binding protein, such as those described in US Publication No. 2007/0178082.

  In some embodiments, a fibronectin based scaffold protein may be bound to a PK moiety comprising a non-proteinaceous polymer. In some embodiments, the polymer is a U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 791, 192 or 4,179,337, polyethylene glycol ("PEG"), polypropylene glycol or polyoxyalkylene. In an exemplary embodiment, the polymer is a PEG moiety.

PEG is a well-known, water-soluble polymer that is commercially available but can also be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sander and Karo, Polymer Synthesis, Academic Press, New). York, Vol. 3, pages 138-161). The term “PEG” is widely used and encompasses any polyethylene glycol molecule, regardless of size, modification at the end of the PEG, and has the formula: X—O (CH ₂ CH ₂ O) _n-1 CH ₂ CH ₂ OH (1) where n is 20 to 2300 and X is H or a terminal modification such as C _1-4 alkyl. In one embodiment, the PEG of the invention terminates at one end with hydroxy or methoxy, ie, X is H or CH ₃ (“methoxy PEG”). PEG may contain additional chemical groups that are necessary for the conjugation reaction; resulting from the chemical synthesis of the molecule; or a spacer for the optimum distance of the parts of the molecule. Further, such PEG can consist of one or more PEG side chains linked together. PEGs having two or more PEG chains are called multi-armed or branched PEGs. Branched PEGs can be prepared by addition of polyethylene oxide to various polyols including, for example, glycerol, pentaerythritol and sorbitol. For example, a 4-arm branched PEG can be prepared from pentaerythritol and ethylene oxide. Branched PEGs are described, for example, in European Published Application No. 473084A and US Pat. No. 5,932,462. One form of PEG includes two PEG side chains (PEG2) linked via the primary amino group of lysine (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62 -69).

  PEG conjugation with a peptide or protein generally involves activation of PEG and coupling of the activated PEG intermediate, either directly with the target protein / peptide or with a linker, which is then Activated and coupled with target protein / peptide (Abuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem., 252, 3582 (1977), Zalipsky , Et al., And Harris et.al., in: Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; . See Chap.21 and 22); New York, 1992: M. Harris ed) Plenum Press. Fibronectin based scaffold proteins containing PEG molecules are also known as conjugated proteins, whereas proteins lacking PEG molecules attached are called unconjugated Note that there are also.

  The size of the PEG utilized will depend on several factors, including the intended use of the fibronectin based scaffold protein. Larger PEGs are preferred for increasing half-life in the body, blood, non-blood extracellular fluids or tissues. For in vivo cellular activity, PEGs in the range of about 10-60 kDa are preferred, as well as PEGs less than about 100 kDa, more preferably less than about 60 kDa, although sizes greater than about 100 kDa can be used as well. For in vivo imaging applications, smaller PEGs, generally less than about 20 kDa, that do not increase half-life to the same extent as large PEGs, but allow for faster distribution and shorter half-life may be used. Various molecular weight forms of PEG can be selected for conjugation to fibronectin based scaffold proteins, eg, about 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300) . The number of repeating units “n” in PEG is estimated relative to the molecular weight described in Daltons. It is preferred that the combined molecular weight of PEG on the activated linker is suitable for pharmaceutical use. Thus, in one embodiment, the molecular weight of the PEG molecule does not exceed 100,000 Da. For example, if 3 PEG molecules are attached to a linker and each PEG molecule has the same molecular weight of 12,000 Da (each n is about 270), the total molecular weight of PEG on the linker is about 36 , 000 Da (total n is about 820). The molecular weight of the PEG attached to the linker may be different, for example, 2 out of 3 molecules on the linker are each 5,000 Da (each n is about 110), one The PEG molecule can be 12,000 Da (n is about 270). In some embodiments, one PEG moiety is conjugated to a fibronectin based scaffold protein. In some embodiments, the PEG moiety is about 20, 30, 40, 50, 60, 70, 80 or 90 KDa. In some embodiments, the PEG moiety is about 40 KDa.

  In some embodiments, a PEGylated fibronectin based scaffold protein comprises one, two or more PEG moieties. In one embodiment, the PEG moiety (s) are bound to amino acid residues on and / or away from the surface of the protein that contacts the target ligand. In one embodiment, the combined molecular weight or total molecular weight of PEG in a PEGylated fibronectin based scaffold protein is from about 3,000 Da to 60,000 Da or from about 10,000 Da to 36,000 Da. In one embodiment, the PEGylated fibronectin based scaffold protein is a substantially linear, linear PEG.

  A person skilled in the art, for example, how a scaffold protein based on PEGylated fibronectin is used therapeutically, the desired dosage, circulation time, resistance to proteolysis, immunogenicity and others Based on the above considerations, a suitable molecular weight of PEG can be selected. For a discussion of PEG and its use to enhance protein properties, see N.C. V. See Katre, Advanced Drug Delivery Reviews 10: 91-114 (1993).

In some embodiments, a fibronectin based scaffold protein uses a poly (ethylene glycol) group —CO (ie, carbonyl) that forms an amide bond to form the formula: —CO— (CH ₂ ) _x —. and one poly (ethylene glycol) group _(OCH 2 _{CH 2)} m -OR, are linked by covalent bond with one of the amino groups of the binding polypeptide; R is lower alkyl; x is 2 or 3 Yes; m is about 450 to about 950; n and m are selected such that the molecular weight of the conjugate minus the binding polypeptide is about 10 to 40 kDa. In one embodiment, the ε-amino group of the lysine of the fibronectin based scaffold protein is an available (free) amino group.

  In one particular embodiment, a carbonate ester of PEG is used to form a PEG-fibronectin based scaffold protein. N, N′-disuccinimidyl carbonate (DSC) may be used in the reaction with PEG to form an active mixed PEG-succinimidyl carbonate, which is then followed by linker determination. It may be reacted with an amino group of a nuclear protein or a fibronectin based scaffold protein (see US Pat. No. 5,281,698 and US Pat. No. 5,932,462). In a similar type of reaction, 1,1 ′-(dibenzotriazolyl) carbonate and di- (2-pyridyl) carbonate are reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed carbonate, respectively. (US Pat. No. 5,382,657).

  PEGylation of fibronectin based scaffold proteins can be performed according to state-of-the-art methods, for example, by reaction of fibronectin based scaffold proteins with electrophilic active PEG (supplier: Shearwater Corp., USA). , Www.shearwatercorp.com). Preferred PEG reagents of the invention include branched N such as, for example, N-hydroxysuccinimidylpropionate (PEG-SPA), butanoate (PEG-SBA), PEG-succinimidylpropionate or mPEG2-NHS. -Hydroxysuccinimide (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69). Such methods may be used to PEGylate the lysine ε-amino group of a fibronectin based scaffold protein or the N-terminal amino group of a fibronectin based scaffold protein.

  In another embodiment, PEG molecules may be coupled to sulfhydryl groups on fibronectin based scaffold proteins (Sartore, L., et al., Appl. Biochem. Biotechnol., 27, 45 (1991). Morpurgo et al., Biocon. Chem., 7, 363-368 (1996); Goodson et al., Bio / Technology (1990) 8, 343; US Pat. No. 5,766,897). US Pat. Nos. 6,610,281 and 5,766,897 describe exemplary reactive PEG species that may be coupled with sulfhydryl groups.

  In some embodiments, PEGylated fibronectin based scaffold proteins are generated by site directed PEGylation, particularly by conjugation of PEG with a cysteine moiety. In certain embodiments, the Cys residue is at the N-terminus, between the N-terminus and the most N-terminal β- or β-like chain, at the C-terminus, or at the C-terminus and most C of the fibronectin-based scaffold protein. Can be located between the ends. In certain embodiments, the fibronectin based scaffold protein is a dimer and the Cys residue is N-terminal and most N-terminal for each binding domain of the fibronectin-based scaffold protein dimer. It may be located between the terminal β or β-like chain, at the C-terminus, or between the C-terminus and the most C-terminus. In certain embodiments, the Cys residue is at the N-terminus of a fibronectin based scaffold protein dimer, of a fibronectin based scaffold protein dimer (ie, a fibronectin based scaffold protein dimer). Between the N-terminus of the N-terminal binding domain and the most N-terminal β- or β-like chain, or at the C-terminus of a fibronectin based scaffold protein dimer, or a fibronectin based scaffold protein dimer Between the C-terminus (ie, of the C-terminal binding domain of a fibronectin-based scaffold protein dimer) and the most C-terminal β or β-like chain. Cys residues may be located in other positions as well, particularly in any of the loops not involved in target binding, or between the binding domains of multivalent fibronectin based scaffold proteins. The PEG moiety may also be attached by other chemical reactions, including by conjugation with amines.

  In some embodiments where the PEG molecule is conjugated to a cysteine residue on a fibronectin based scaffold protein, the cysteine residue is native to the fibronectin based scaffold protein, whereas In other embodiments, one or more cysteine residues are engineered in a fibronectin based scaffold protein. Mutations may be introduced into sequences encoding fibronectin based scaffold proteins to generate cysteine residues. This can be accomplished, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutating to cysteine residues include serine, threonine, alanine and other hydrophilic residues. The residue that is mutated to cysteine is preferably a residue that is exposed to the surface. Algorithms for predicting surface accessibility of residues based on primary sequence or protein are well known in the art. Alternatively, comparing the amino acid sequence of the fibronectin-based scaffold protein, given that the crystal structure of the 10fn3 domain framework, on which the fibronectin-based scaffold protein has been designed, has been solved Can predict surface residues (see Himanen et al., Nature. (2001) 20-27; 414 (6866): 933-8), thus identifying residues exposed to the surface. Is done. In one embodiment, cysteine residues are introduced into fibronectin based scaffold proteins at or near the N and / or C terminus, or within the loop region. PEGylation of cysteine residues can be performed using, for example, PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide or PEG-orthopyridyl disulfide.

  In some embodiments, the PEGylated fibronectin based scaffold protein comprises a PEG molecule covalently linked to the α-amino group of the N-terminal amino acid. Site-specific N-terminal reductive amination is described in Pepinsky et al. (2001) JPETE, 297, 1059 and US Pat. No. 5,824,784. The use of PEG-aldehydes for reductive amination of proteins utilizing other available nucleophilic amino groups is described in US Pat. No. 4,002,531, Wieder et al. (1979) J. MoI. Biol. Chem. 254, 12579, and Chamow et al. (1994) Bioconjugate Chem. 5, 133.

  In another embodiment, the PEGylated fibronectin based scaffold protein comprises one or more PEG molecules covalently linked to a linker, which is then at the N-terminus of the fibronectin based scaffold protein. It is combined with the α-amino group of the amino acid residue. Such an approach is disclosed in US Publication No. 2002/0044921 and International Publication No. 94/01451.

  In one embodiment, the fibronectin based scaffold protein is PEGylated at the C-terminus. In certain embodiments, the protein is PEGylated at the C-terminus by introduction of a C-terminal azido-methionine followed by conjugation of a methyl-PEG-triarylphosphine compound via a Staudinger reaction. This C-terminal conjugation method is described in Cazalis et al. , C-Terminal Site-Specific PEGylation of a Truncated Thrombomodulin Mutant with Retention of Full Bioactivity, Bioconjug Chem. 2004; 15 (5): 1005-1009.

  In an exemplary embodiment, a fibronectin based scaffold protein is PEGylated at the C-terminal tail region as further described herein. Exemplary C-terminal tails include, for example, a polypeptide having any one of SEQ ID NOs: 5, 6 or 35.

  Conventional separation and purification techniques known in the art such as size exclusion (eg gel filtration) and ion exchange chromatography can be used to purify scaffold proteins based on PEGylated fibronectin. Products may be separated using SDS-PAGE. Separable products include mono-, di-, tri-, polyPEGylated and non-PEGylated fibronectin based scaffold proteins, and free PEG. The percentage of mono PEG conjugate can be controlled by pooling a wide fraction around the elution peak to increase the percentage of mono PEG in the composition. About 90 percent of the monoPEG conjugate represents a good balance between yield and activity. For example, a composition in which at least 92 percent or at least 96 percent of the conjugate is a monoPEG species may be desirable. In an embodiment of the invention, the percentage of monoPEG conjugate is from 90 percent to 96 percent.

  In one embodiment of the invention, PEG in a PEGylated fibronectin based scaffold protein uses a hydroxylamine assay, eg, 450 mM hydroxylamine (pH 6.5) at room temperature for 8 to 16 hours. Thus, it is not hydrolyzed from PEGylated amino acid residues and is therefore stable. In one embodiment, more than 80% of the composition is a scaffold protein based on a stable monoPEG fibronectin, more preferably at least 90%, and most preferably at least 95%.

In another embodiment, the PEGylated fibronectin based scaffold protein is at least about 25%, 50%, 60%, 70%, 80%, 85%, 90% of the biological activity associated with the unmodified protein. , 95% or 100% is preferred. In one embodiment, the biological activity, K _D, is evaluated by the k _on or k _off, it refers to its ability to bind to one or more target molecules. In one specific embodiment, a PEGylated fibronectin based scaffold protein exhibits an increase in binding to one or more target molecules compared to a non-PEGylated fibronectin based scaffold protein.

The serum clearance rate of scaffold proteins based on PEG-modified fibronectin is about 10%, 20%, 30%, 40%, 50% compared to the clearance rate of unmodified fibronectin based scaffold proteins. 60%, 70%, 80% or even 90%. A scaffold protein based on PEG-modified fibronectin can have an enhanced half-life (t _1/2 ) compared to the half-life of an unmodified fibronectin-based scaffold protein. The half-life of the scaffold protein based on PEG-modified fibronectin is at least 10%, 20%, 30%, 40%, 50% compared to the half-life of the scaffold protein based on unmodified fibronectin. It can be enhanced to 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even 1000%. In some embodiments, the protein half-life is determined in vitro, such as in buffered saline or serum. In other embodiments, the protein half-life is an in vivo half-life, such as the half-life of a fibronectin based scaffold protein in animal serum or other bodily fluids.

Nucleic acid-protein fusion technology In one aspect, the application is based on fibronectin comprising a fibronectin type III domain that binds to a human target, such as, for example, TNF-α, EGFR, VEGFR2, IGF-IR, or other proteins. Provide scaffold protein. One method for rapidly creating and testing Fn3 domains with specific binding properties is the nucleic acid-protein fusion technology of the Adnexus, Bristol-Myers Squibb company. Novel polypeptides and amino acids that are important for protein binding using such in vitro expression and tag technology, referred to as the PROfusion ^trademark , which utilizes nucleic acid-protein fusions (RNA- and DNA-protein fusions) Motifs can be identified. Nucleic acid-protein fusion technology is a technology for covalently coupling a protein to its encoded genetic information. For a detailed description of RNA-protein fusion techniques and fibronectin-based scaffold protein library screening methods, see Szostak et al., Incorporated herein by reference. U.S. Pat. Nos. 6,258,558; 6,261,804; 6,214,553; 6,281,344; 6,207,446; , 518,018; International Publication No. WO 00/34784; International Publication No. WO 01/64942; International Publication No. WO 02/032925; and Roberts and Szostak, Proc Natl. Acad. Sci. 94: 12297-122302, 1997.

Vectors & Polynucleotides Nucleic acids encoding any of the various fibronectin based scaffold proteins disclosed herein may be synthesized chemically, enzymatically or recombinantly. The codon usage can be selected to improve expression in the cell. Such codon usage varies depending on the cell type selected. For E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells, specialized codon usage patterns have been developed. For example: Mayfieldd et al. Proc Natl Acad Sci US 2003 Jan 21; 100 (2): 438-42; Sinclair et al. Protein Expr Purif. 2002 Oct; 26 (1): 96-105; Connell ND. Curr Opin Biotechnol. 2001 Oct; 12 (5): 446-9; Makrides et al. Microbiol Rev. 1996 Sep; 60 (3): 512-38; and Sharp et al. Yeast. 1991 Oct; 7 (7): 657-78.

  General techniques for nucleic acid manipulation are described, for example, in Sambrook et al., Incorporated herein by reference. , Molecular Cloning ,: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed. , 1989 or F.R. Ausubel et al. , Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and periodic updates. The DNA encoding the polypeptide is operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, viral or insect genes. Such regulatory elements include a transcriptional promoter, any operator sequence to control transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls termination of transcription and translation. The ability to replicate in the host is usually conferred by the origin of replication and further incorporates a selection gene that facilitates recognition of the transformant.

  The fibronectin based scaffold proteins described herein are not only directly, but are preferably signal sequences or mature proteins or other polypeptides having a specific cleavage site at the N-terminus of the polypeptide. A fusion polypeptide with a heterologous polypeptide may also be produced recombinantly. The heterologous signal sequence selected is preferably one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence, the signal sequence is replaced with, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or a thermostable enterotoxin II leader. For yeast secretion, natural signal sequences can be derived from, for example, yeast invertase leaders, factor leaders (including Saccharomyces and Kluyveromyces alpha factor leaders) or acid phosphatase leaders, C.I. Substitution may be by the signals described in the albicans glucoamylase leader or PCT WO 90/13646. For mammalian cell expression, mammalian signal sequences as well as viral secretory leaders such as herpes simplex gD signals are available. Such precursor region DNA may be ligated in reading frame to DNA encoding the protein.

  Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. In general, for cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, a 2 micron plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are mammalian. Useful for cloning vectors in cells. In general, an origin of replication component is not required for mammalian expression vectors (the SV40 origin is usually used simply because it contains the early promoter).

  Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes are (a) conferring resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophy deficiencies, or (c) from complex media It encodes a protein that supplies critical nutrients that are not available, such as the gene encoding the D. alanine racemase of Bacilli.

  A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). The trp1 gene provides a selectable marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1 lesion in the yeast host cell genome thus provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids carrying the Leu2 gene.

  Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding a fibronectin based scaffold protein. Suitable promoters for use with prokaryotic hosts include hybrid promoters such as the phoA promoter, β-lactamase and lactose promoter system, alkaline phosphatase, tryptophan (trp) promoter system, and tac promoter. However, other known bacterial promoters are also suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to DNA encoding a fibronectin based scaffold protein.

  Promoter sequences for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of a large number of genes is a CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence that can be a signal for the addition of a poly A tail to the 3' end of the coding sequence. All of these sequences are inserted appropriately into eukaryotic expression vectors.

  Examples of facilitating sequences suitable for use with yeast hosts include 3-phosphoglycerate kinase, or enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6 -Promoters for other glycolytic enzymes such as phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase and glucokinase.

  Other yeast promoters, which are inducible promoters with the additional advantage of transcription controlled by growth conditions, include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradation enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde-3 There is a promoter region for phosphate dehydrogenase and enzymes involved in maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in European Patent Publication No. 73,657. Yeast enhancers are also advantageously used with yeast promoters.

  Transcription from vectors in mammalian host cells is, for example, polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and Most preferably, such a promoter is isolated from the host cell system by a promoter derived from the genome of a virus such as simian virus 40 (SV40), from a heterologous mammalian promoter, such as an actin promoter or an immunoglobulin promoter, and from a heat shock promoter. If fit, it can be controlled.

  The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in US Pat. No. 4,419,446. A modification of this system is described in US Pat. No. 4,601,978. For the expression of human β-interferon cDNA in mouse cells under the control of the herpes simplex virus-derived thymidine kinase promoter, see Reyes et al. , Nature 297: 598-601 (1982). Alternatively, a rous sarcoma virus long terminal repeat can be used as a promoter.

  Transcription of DNA encoding fibronectin-based scaffold proteins by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. A number of enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Usually, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer behind the origin of replication (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297: 17-18 (1982) on enhancer elements for activation of eukaryotic promoters. Enhancers can be spliced into the vector at positions 5 'or 3' of the polypeptide coding sequence, but are preferably located 5 'to the promoter.

  Expression vectors used in eukaryotic host cells (eg, nucleated cells from yeast, fungi, insects, plants, animals, humans or other multicellular organisms) also stabilize mRNA and terminate transcription. It will also contain the sequences necessary to do. Such sequences can generally be obtained from the 5 'untranslated region and optionally the 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector disclosed therein.

  The recombinant DNA may also include any type of protein tag sequence that may be useful for purifying fibronectin based scaffold proteins. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags or GST tags. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts can be found in Cloning Vector: A Laboratory Manual, (Elsevier, New York, 1985), the relevant disclosures of which are , Incorporated herein by reference.

  The expression construct is introduced into the host cell using methods appropriate for the host cell, as will be apparent to those skilled in the art. Various methods for introducing nucleic acids into host cells are known in the art and include electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other materials; microparticle guns; lipofection And (but is not limited to) infection (where the vector is an infectious agent).

  Suitable host cells include prokaryotes, yeast, mammalian cells or bacterial cells. Suitable bacteria include gram negative or gram positive bacteria such as E. coli or Bacillus species. Preferably, S.M. Yeasts derived from Saccharomyces species, such as cerevisiae, may also be used for the production of polypeptides. Various mammalian or insect cell culture systems can also be used to express recombinant protein. Baculovirus systems for producing heterologous proteins in insect cells are reviewed in Luckow and Summers, (Bio / Technology, 6:47, 1988). Examples of suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T and A BHK cell line is mentioned. Purified fibronectin based scaffold proteins are prepared by culturing a suitable host / vector system to express the recombinant protein. For many applications, the small size fibronectin based scaffold proteins disclosed herein are expressed in E. coli as a preferred method for expression. The fibronectin based scaffold protein is then purified from the culture medium or cell extract.

Protein production Host cells are transformed with the expression or cloning vectors described herein for protein production to induce a promoter, select transformants, or a gene encoding the desired sequence. It is cultured in a conventional nutrient medium suitably modified for amplification.

Host cells used to produce fibronectin based scaffold proteins may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential media ((MEM), (Sigma)), RPMI-1640 (Sigma) and Dulbecco's modified Eagle's media ((DMEM), (Sigma)) are host cells. Suitable for culturing. Furthermore, Ham et al. , Meth. Enz. 58:44 (1979), Barnes et al. , Anal. Biochem. 102: 255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122. 469; International Publication No. 90/03430; International Publication No. 87/00195; or US Pat. No. Re. Any of the media described in US Pat. No. 30,985 may be used as a culture medium for host cells. Any of these media may contain hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), as needed. Supplemented with nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ^trademark ), trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar range) and glucose or equivalent energy source obtain. Any other necessary nutritional supplements may be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc., have been used so far with the host cell selected for expression and will be apparent to those skilled in the art.

  The fibronectin based scaffold proteins disclosed herein can also be produced using a cell-free translation system. For these purposes, nucleic acids encoding fibronectin-based scaffold proteins enable in vitro transcription to produce mRNA and can be used to identify the specific cell-free system used (eukaryotic cells such as mammalian or yeast cells). It must be modified to allow cell-free translation of mRNA in translation systems that do not contain or translation systems that do not contain prokaryotes such as bacterial cells).

  Fibronectin based scaffold proteins can also be produced by chemical synthesis (eg, by the method described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, IL). Modifications to fibronectin based scaffold proteins can also be generated by chemical synthesis.

  Fibronectin based scaffold proteins can be purified by protein isolation / purification methods commonly known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (eg, using ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, sequential Phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combination thereof may be mentioned. After purification, the fibronectin based scaffold protein may be exchanged for a different buffer by any of a variety of methods known in the art, including but not limited to filtration and dialysis. And / or may be concentrated.

  The purified fibronectin based scaffold protein is preferably at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact figure of purity, fibronectin based scaffold proteins are sufficiently pure for use as pharmaceutical formulations.

Exemplary Uses In one aspect, the application provides a fibronectin based scaffold protein labeled with a detectable moiety. Fibronectin based scaffold proteins may be used for various diagnostic applications. The detectable moiety can be anything that can produce a detectable signal either directly or indirectly. For example, the detectable moiety may be a radioisotope such as H3, C14, C13, P32, S35 or I131; a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine or luciferin; or alkaline phosphatase, β-galactosidase or horseradish It can be an enzyme such as peroxidase.

  Hunter, et al. , Nature 144: 945 (1962); David, et al. Biochemistry 13: 1014 (1974); Pain, et al. , J. et al. Immunol. Meth. 40: 219 (1981); and Nygren, J. et al. Histochem. and Cytochem. 30: 407 (1982), any method known in the art for conjugating proteins to detectable moieties may be used. In vitro methods include conjugation chemistry well known in the art, including chemical reactions compatible with proteins, such as chemical reactions for specific amino acids such as Cys and Lys. In order to link the detectable moiety to a fibronectin based scaffold protein, a linking group or reactive group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photosensitive groups, peptidase labile groups and esterase labile groups. Preferred linking groups include disulfide groups and thioether groups depending on the application. For polypeptides that do not contain Cys amino acids, Cys may be engineered into a position that allows the activity of the protein to be present while creating a position for conjugation.

  Fibronectin based scaffold proteins linked by a detectable moiety are also useful for in vivo imaging. The polypeptide can be linked to a subject, preferably a radio-opaque substance or radioisotope administered in the bloodstream, and the presence and location of the labeled protein in the subject is assayed. . This imaging technique is useful, for example, in the classification and treatment of malignant tumors when fibronectin based scaffold proteins bind to cancer-related targets. Fibronectin based scaffold proteins may be labeled with any moiety that is detectable in a subject, whether by nuclear magnetic resonance, radiology, or by other detection means known in the art. .

  Fibronectin based scaffold proteins are also useful as affinity purification substances. In this process, the fibronectin based scaffold protein is immobilized on a suitable support such as Sephadex resin or filter paper using methods well known in the art.

  Fibronectin-based scaffold proteins can be used in any known assay method, such as competitive binding experiments, direct and indirect sandwich assays and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Technologies, pp. 147-158). (CRC Press, Inc., 1987)).

  In certain aspects, the present disclosure provides a method of detecting a target molecule in a sample such as VEGFR2, IGF-IR or EGFR. The method comprises contacting a sample with a fibronectin based scaffold protein as described herein, wherein said contacting is under conditions that allow fibronectin based scaffold protein-target complex formation. And detecting the complex, thereby detecting the target in the sample. Detection may be performed using any technique known in the art such as, for example, radiography, immunological assay, fluorescence detection, mass spectrometry or surface plasmon resonance. The sample is often a biological sample, such as a biopsy, particularly a tumor, biopsy of a suspicious tumor. The sample can be from a human or other mammal. Fibronectin based scaffold proteins can be labeled with a labeling moiety such as a radioactive moiety, a fluorescent moiety, a chromogenic moiety, a chemiluminescent moiety or a hapten moiety. Fibronectin based scaffold proteins can be immobilized on a solid support.

In one aspect, the application provides fibronectin based scaffold proteins that are useful in the treatment of disorders. The disease or disorder that can be treated will depend on the binding specificity of the fibronectin based scaffold protein. The application also provides a method of administering a fibronectin based scaffold protein to a subject. In some embodiments, the subject is a human. In some embodiments, fibronectin based scaffold proteins are pharmaceutically acceptable to mammals, particularly humans. A “pharmaceutically acceptable” polypeptide refers to a polypeptide that is administered to an animal without appreciable adverse medical consequences. The pharmaceutically acceptable fibronectin Examples of scaffold proteins based free of ^{10 Fn3} domain and essentially endotoxin lack integrin-binding domain (RGD), or include ^{10 Fn3} domains with very low endotoxin levels.

  In certain embodiments, fibronectin based scaffold proteins, particularly fibronectin based scaffold proteins that bind to IGF-IR, VEGFR2 and / or EGFR are useful in the treatment of disorders such as cancer. In certain embodiments, fibronectin based scaffold proteins are useful in the treatment of cancer disorders associated with IGF-IR, VEGFR2 and / or EGFR mutations or expression levels. In some embodiments, administration of the fibronectin based scaffold protein treats an antiproliferative disorder in the subject. In some embodiments, administration of a fibronectin based scaffold protein inhibits tumor cell growth in vivo. Tumor cells can be derived from any cell type including, but not limited to, epidermis, epithelium, endothelium, leukemia, sarcoma, multiple myeloma or mesoderm cells. Examples of common tumor cell lines for use in xenograft tumor studies include A549 (non-small cell lung cancer) cells, DU-145 (prostate) cells, MCF-7 (breast) cells, Colo205 (colon) Cells, 3T3 /] GF-IR (mouse fibroblast) cells, NCI H441 cells, HEP G2 (hepatoma) cells, MDA MB231 (breast) cells, HT-29 (colon) cells, MDA-MB-435s ( Breast) cells, U266 cells, SH-SY5Y cells, Sk-Mel-2 cells, NCI-H929, RPM18226 and A431 cells. In some embodiments, a fibronectin based scaffold protein inhibits tumor cell growth as compared to tumor growth in an untreated animal. In some embodiments, fibronectin based scaffold proteins inhibit tumor cell growth by 50, 60, 70, 80% or more compared to tumor growth in untreated animals. In some embodiments, inhibition of tumor cell growth is measured at least 7 days or at least 14 days after the animal initiates a fibronectin based scaffold protein. In some embodiments, another antineoplastic agent is administered to the animal along with a fibronectin based scaffold protein.

  In certain aspects, the present disclosure provides a method of administering a fibronectin based scaffold protein for the treatment and / or prevention of tumors and / or tumor metastases, wherein the tumor is a brain tumor, urogenital Particularly preferred is selected from the group consisting of tract tumor, lymphoid tumor, stomach tumor, pharyngeal tumor, monocytic leukemia, lung adenocarcinoma, small cell lung carcinoma, pancreatic cancer, glioblastoma and breast cancer Not limited to that.

  In certain aspects, the present disclosure provides a fibronectin based scaffold protein for squamous cell carcinoma, bladder cancer, gastric cancer, liver cancer, kidney cancer, colorectal cancer, breast cancer, head cancer, cervical cancer, esophageal cancer, Methods of administration are provided for the treatment of cancerous diseases selected from the group consisting of gynecological cancer, thyroid cancer, lymphoma, chronic leukemia and acute leukemia.

  In other embodiments, a fibronectin based scaffold protein binds to a target involved in inflammatory reactions and / or autoimmune diseases, such as, for example, tumor necrosis factor (TNF) α. Such fibronectin based scaffold proteins may be useful for treating autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and refractory asthma.

Formulation and Administration The application further provides a pharmaceutically acceptable composition comprising a fibronectin based scaffold protein as described herein, wherein the composition is essentially free of endotoxin. .

A therapeutic formulation comprising a fibronectin based scaffold protein is prepared by mixing the described protein having the desired degree of purity with an optional physiologically acceptable carrier, excipient or stabilizer. , Lyophilized, or in the form of other dried formulations, prepared for storage (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used, buffers such as phosphate, citrate and other organic acids; ascorbic acid and methionine Preservatives (octadecydimethylbenzyloxychloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol Resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; such as serum albumin, gelatin or immunoglobulin Hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextran; chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and / or non-ions such as TWEEN ^trademark , PLURONICS ^trademark or polyethylene glycol (PEG) Surfactants.

  The formulation herein may also include more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combinations of amounts that are effective for the purpose intended.

  Fibronectin based scaffold proteins can also be prepared in microcapsules prepared by, for example, coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methyl methacrylate) microcapsules, respectively. May be encapsulated in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. et al. Ed. (1980).

  In certain embodiments, the application provides a stable composition of fibronectin based scaffold proteins having a pH of 4.0-6.5. In other embodiments, the application provides a stable composition of fibronectin based scaffold proteins having a pH of 4.0-5.5. In another embodiment, the application provides a stable composition of fibronectin based scaffold protein having a pH of 5.5. In another embodiment, the application provides a stable composition of a fibronectin based scaffold protein having a pH of 4.0. In particular, the application provides stable compositions of fibronectin based scaffold proteins that have reduced fragmentation and / or low levels of aggregation during storage in solution. As demonstrated in the exemplary section, the fibronectin based scaffold proteins described herein have increased stability at pH 4.0, while at the same time compared to pH 5.5. It showed a reduced aggregation level at pH 4.0. Such a stable and soluble formulation having a pH of 4.0 is particularly suitable for intravenous administration. In some embodiments, the protein concentration in such stable formulations is at least 3 mg / mL. In an exemplary embodiment, the protein concentration in such stable formulations is at least 5 mg / mL. In certain embodiments, the protein concentration in such stable formulations is 3-10 mg / mL, 3-8 mg / mL, 3-6 mg / mL, 3-5 mg / mL, 4-10 mg / mL, 4-8 mg / ML, 4-6 mg / mL, 5-10 mg / mL, 5-8 mg / mL or 5-6 mg / mL. In an exemplary embodiment, a stable composition of fibronectin based scaffold protein has reduced aggregation compared to an equivalent formulation of fibronectin based scaffold protein at higher pH. . For example, a stable formulation can be obtained in a 4 week solution at pH 4.0 compared to the aggregation level seen during storage of fibronectin-based scaffold proteins during storage for 4 weeks at pH 5.5 and above. During storage, it may exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or less aggregation. In certain embodiments, a stable formulation of fibronectin based scaffold protein is 10%, 8%, 7%, 6%, 5%, 4%, 3% after storage at 25 ° C. for at least 4 weeks. %, 2%, 1% or less agglomeration. In certain embodiments, a stable formulation of a fibronectin based scaffold protein is 7%, 6%, 5%, 4%, 3%, stored in solution at pH 4.0 and 25 ° C. for 4 weeks. It has 5%, 3%, less than 2% or less fragmentation. In certain embodiments, a stable formulation of a fibronectin based scaffold protein has less than 5% fragmentation and less than 5% aggregation during storage in solution at 25 ° C. for at least 4 weeks. In an exemplary embodiment, a stable formulation of a fibronectin based scaffold protein has less than 4% fragmentation and less than 4% aggregation during storage in solution at 25 ° C. for at least 4 weeks. .

  Formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes.

Sustained release formulations may be prepared. Suitable examples of sustained release formulations include solid hydrophobic polymer semipermeable matrices containing fibronectin based scaffold proteins as described herein, wherein the matrix is a molded article, for example, It is in the form of a film or microcapsule. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L-glutamic acid and yethyl. Degradable lactic acid-glycolic acid copolymers such as L-glutamine copolymer, non-degradable ethylene-vinyl acetate, LUPRON DEPOT ^trademark (injectable microspheres comprising lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-)-3-hydroxybutyric acid is mentioned. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow release of molecules over 100 days, certain hydrogels release proteins for shorter periods of time. If the encapsulated proteins of the invention remain in the body for a long time, they denature or aggregate as a result of exposure to humidity at 37 ° C, resulting in loss of biological activity and possible changes in immunogenicity. obtain. Depending on the mechanism involved, a rational strategy for stabilization can be devised. For example, if the aggregation mechanism is found to be intermolecular S—S bond formation by thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solution, reducing moisture content. It can be achieved by controlling, using appropriate additives and developing specific polymer matrix compositions.

  One skilled in the art will appreciate that the dosage of each fibronectin based scaffold protein will vary depending on the identity of the protein, but a preferred dosage is from about 10 mg / square meter to about 2000 mg / square meter, more preferably , Ranging from about 50 mg / square meter to about 1000 mg / square meter.

  For therapeutic applications, fibronectin based scaffold proteins are administered to a subject in a pharmaceutically acceptable dosage form. They can be administered as a bolus or intravenously by continuous infusion over a period of time, intramuscular, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation route. Proteins can also be administered by intratumoral, peritumoral, intralesional or perilesional routes and exert local as well as systemic therapeutic effects. Suitable pharmaceutically acceptable carriers, diluents and excipients are well known and can be determined by one skilled in the art as the clinical situation requires. Examples of suitable carriers, diluents and / or excipients: (1) Dulbecco's phosphate buffered saline containing about 1 mg / ml to 25 mg / ml human serum albumin, pH about 7.4, (2) 0.9% saline (0.9% w / v NaCl) and (3) 5% (w / v) dextrose. The methods of the invention can be performed in vitro, in vivo, or ex vivo.

  Administration of the fibronectin based scaffold protein and one or more additional therapeutic agents can occur as described above for therapeutic applications, whether coadministered or sequentially administered. It is understood that pharmaceutically acceptable carriers, diluents and excipients suitable for co-administration will vary depending on the identity of the particular therapeutic agent being co-administered by those skilled in the art.

  Fibronectin based scaffold proteins are usually formulated at concentrations of about 0.1 mg / ml to 100 mg / ml when present in an aqueous dosage form rather than lyophilized. Wide variations outside the range are acceptable. Suitable dosages of fibronectin based scaffold protein for the treatment of disease include the type of disease being treated, severity and course of the disease, fibronectin based scaffold protein as defined above Is administered for prophylactic or therapeutic purposes, depending on the course of treatment so far, the patient's medical history and response to fibronectin-based scaffold proteins, and the discretion of the attending physician. A fibronectin based scaffold protein is suitably administered to a patient at one time or over a series of treatments.

ＤＫ＋ＥＧＦＲ／ＩＧＦ−ＩＲバインダー（配列番号：２３）

ＧＳＧＣテールを備えるＤＫ−ＥＧＦＲ／ＩＧＦ−ＩＲバインダー（配列番号：２４）

ＥＩＥＫＰＣＱテールを備えるＤＫ−ＥＧＦＲ／ＩＧＦ−ＩＲバインダー（配列番号：２５）

Ｘ_ｎＳＤＶＰＲＤＬ， ｎ＝０、１または２アミノ酸であり、ｎ＝１の場合、ＸはＭｅｔまたはＧｌｙであり、ｎ＝２の場合、ＸはＭｅｔ−Ｇｌｙである（配列番号：２６）

Ｘ_ｎＤＶＰＲＤＬ， ｎ＝０、１または２アミノ酸であり、ｎ＝１の場合、ＸはＭｅｔまたはＧｌｙであり、ｎ＝２の場合、ＸはＭｅｔ−Ｇｌｙである（配列番号：２７）

Ｘ_ｎＶＰＲＤＬ， ｎ＝０、１または２アミノ酸であり、ｎ＝１の場合、ＸはＭｅｔまたはＧｌｙであり、ｎ＝２の場合、ＸはＭｅｔ−Ｇｌｙである（配列番号：２８）

Ｘ_ｎＰＲＤＬ， ｎ＝０、１または２アミノ酸であり、ｎ＝１の場合、ＸはＭｅｔまたはＧｌｙであり、ｎ＝２の場合、ＸはＭｅｔ−Ｇｌｙである（配列番号：２９）

Ｘ_ｎＲＤＬ， ｎ＝０、１または２アミノ酸であり、ｎ＝１の場合、ＸはＭｅｔまたはＧｌｙであり、ｎ＝２の場合、ＸはＭｅｔ−Ｇｌｙである（配列番号：３０）

Ｘ_ｎＤＬ， ｎ＝０、１または２アミノ酸であり、ｎ＝１の場合、ＸはＭｅｔまたはＧｌｙであり、ｎ＝２の場合、ＸはＭｅｔ−Ｇｌｙである（配列番号：３１）

ＥＩＥＫＰＳＱ（配列番号：３２）
ＥＩＥＫＰ（配列番号：３３）
ＥＩＥＫＰＳ（配列番号：３４）
ＥＩＥＫＰＣ（配列番号：３５）
ＥＧＳＧＳ（配列番号：３６）

ＷＴフィブロネクチン配列

ＥＩＥＫテールを備える^１０Ｆｎ３コア
ＥＶＶＡＡＴＰＴＳＬＬＩＳＷ（Ｘ）_ｘＲＹＹＲＩＴＹＧＥＴＧＧＮＳＰＶＱＥＦＴＶＰ（Ｘ）_ｙＴＡＴＩＳＧＬＫＰＧＶＤＹＴＩＴＶＹＡＶＴ（Ｘ）_ｚＰＩＳＩＮＹＲＴＥＩＥＫ（配列番号：３８）

Ｅコア（配列番号：３９）
ＥＶＶＡＡＴＰＴＳＬＬＩＳＷＷＡＰＶＤＲＹＱＹＹＲＩＴＹＧＥＴＧＧＮＳＰＶＱＥＦＴＶＰＲＤＶＹＴＡＴＩＳＧＬＫＰＧＶＤＹＴＩＴＶＹＡＶＴＤＹＫＰＨＡＤＧＰＨＴＹＨＥＳＰＩＳＩＮＹＲＴ

ＩＧＦ−ＩＲ ＢＣループ（配列番号：４０）
ＳＡＲＬＫＶＡ

ＩＧＦ−ＩＲ ＤＥループ（配列番号：４１）
ＫＮＶＹ

ＩＧＦ−ＩＲ ＦＧループ（配列番号：４２）
ＲＦＲＤＹＱ

ＶＥＧＦＲ２ ＢＣループ（配列番号：４３）
ＲＨＰＨＦＰＴ

ＶＥＧＦＲ２ ＤＥループ（配列番号：４４）
ＬＱＰＰ

ＶＥＧＦＲ２ ＦＧループ（配列番号：４５）
ＤＧＲＮＧＲＬＬＳＩ

ＥＩＤＫ（配列番号：４６）

ＥＩＤＫＰＣＱ（配列番号：４７）

Ｃｙｓテールを備えるＩ−Ｆｎ−Ｖ（２ＤＫ−）（配列番号：４８）

ｓｅｒテールを備えるＩ−Ｆｎ−Ｖ（２ＤＫ−）（配列番号：４９）

ｓｅｒまたはｃｙｓテールを備えるＩ−ＧＳ５−Ｖ（２ＤＫ−）（配列番号：５０）

ｓｅｒまたはｃｙｓテールを備えるＩ−ＧＳ１０−Ｖ（２ＤＫ−）（配列番号：５１）

ｓｅｒテールを備えるＶ−Ｆｎ−Ｉ（２ＤＫ−）（配列番号：５２）

ｃｙｓテールを備えるＶ−Ｆｎ−Ｉ（２ＤＫ−）（配列番号：５３）

ｓｅｒまたはｃｙｓテールを備えるＶ−ＧＳ５−Ｉ（２ＤＫ−）（配列番号：５４）

ｓｅｒまたはｃｙｓテールを備えるＶ−ＧＳ１０−Ｉ（２ＤＫ−）（配列番号：５５）

ＶＩ（ＤＫ＋）（配列番号：５６）

ＶＩ（ＤＫ−）（配列番号：５７）

Sequence Listing WT Core Array EVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTSTATISGLKPGVDYTITVYAVTGRDSPASSKPISINYRT (SEQ ID NO: 1)

I core (SEQ ID NO: 2)
EVVAATPTSLLISWSARLKVARYYRITGGETGSGNSPVQEFTVPKNVYTATIGLKPGVDYTITVYAVTRFRDYQPISINYRT

V core (SEQ ID NO: 3)
EVVAATPTSLLISWRHFPFPRYYRIGHTYGETGGNSPVQEFTVPLQPPTATIGLLKPGVDYTITVYAVTDGRNGLRLLSIPISINYRT

Short tail (SEQ ID NO: 4)
EIEK

Modified Cys tail (SEQ ID NO: 5)
EGSGC

Cys tail (SEQ ID NO: 6)
EIEKPCQ

Fn-based linker (SEQ ID NO: 7)
PSTSTST

GS ₅ linker (SEQ ID NO: 8)
GSGSGSGSGS

GS ₁₀ linker (SEQ ID NO: 9)
GSGSGSGSGSGSGSGSGSGS

(GGGGS) ₃ (SEQ ID NO: 10)
GGGGS GGGGS GGGGS

(GGGGS) ₅ (SEQ ID NO: 11)
GGGGS GGGGS GGGGS GGGGS GGGGS

G ₄ SG ₄ SG ₃ SG (SEQ ID NO: 12)
GGGGSGGGGSGGGSGG

GPG (SEQ ID NO: 13)

GPGPGPG (SEQ ID NO: 14)

GPGPGPGPGPG (SEQ ID NO: 15)

PA3 linker (SEQ ID NO: 16)
PAPAPA

PA6 linker (SEQ ID NO: 17)
PAPAPAPAPAPAPA

PA9 linker (SEQ ID NO: 18)
PAPAPAPAPAPAPAPAPAPA

MGVSDVPPRDL (SEQ ID NO: 19)

VSDVPPRDL (SEQ ID NO: 20)

GVSDVPPRDL (SEQ ID NO: 21)

DK + VEGFR2 / IGF-IR binder (SEQ ID NO: 22)

DK + EGFR / IGF-IR binder (SEQ ID NO: 23)

DK-EGFR / IGF-IR binder with GSGC tail (SEQ ID NO: 24)

DK-EGFR / IGF-IR binder with EIEKPCQ tail (SEQ ID NO: 25)

X _n SDVPRDL, a n = 0, 1 or 2 amino acids in the case of n = 1, X is Met or Gly, for n = 2, X is Met-Gly (SEQ ID NO: 26)

X _n DVPRDL, n = 0, 1 or 2 amino acids, when n = 1, X is Met or Gly, when n = 2, X is Met-Gly (SEQ ID NO: 27)

X _n VPRDL, n = 0, 1 or 2 amino acids, when n = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 28)

X _n PRDL, n = 0, 1 or 2 amino acids, when n = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 29)

X _n RDL, n = 0, 1 or 2 amino acids, when n = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 30)

X _n DL, a n = 0, 1 or 2 amino acids in the case of n = 1, X is Met or Gly, for n = 2, X is Met-Gly (SEQ ID NO: 31)

EIEKPSQ (SEQ ID NO: 32)
EIEKP (SEQ ID NO: 33)
EIEKPS (SEQ ID NO: 34)
EIEKPC (SEQ ID NO: 35)
EGSGS (SEQ ID NO: 36)

WT fibronectin sequence

¹⁰ Fn3 core EVVAATPTSLLISW (X) with EIEK tail _x RYYRITYYGETGGNSPVQEFTVP (X) _y TATIGLSKPGVDYTITVYAVT (X) _z PISINYRTEIEK (SEQ ID NO: 38)

E core (SEQ ID NO: 39)
EVVAATPTSLLISWWWAPVDRYQYYRITYGETGGNSPVQEFTVPRDVYTATIGLSKPGVDYTITVYAVVTDYKPHADGPTYHESPISINYRT

IGF-IR BC loop (SEQ ID NO: 40)
SARLKVA

IGF-IR DE loop (SEQ ID NO: 41)
KNVY

IGF-IR FG loop (SEQ ID NO: 42)
RFRDYQ

VEGFR2 BC loop (SEQ ID NO: 43)
RHPHFPT

VEGFR2 DE loop (SEQ ID NO: 44)
LQPP

VEGFR2 FG loop (SEQ ID NO: 45)
DGRNGRLLSI

EIDK (SEQ ID NO: 46)

EIDKPCQ (SEQ ID NO: 47)

I-Fn-V (2DK-) with Cys tail (SEQ ID NO: 48)

I-Fn-V (2DK-) with ser tail (SEQ ID NO: 49)

I-GS5-V (2DK-) with a ser or cys tail (SEQ ID NO: 50)

I-GS10-V (2DK-) with ser or cys tail (SEQ ID NO: 51)

V-Fn-I (2DK-) with ser tail (SEQ ID NO: 52)

V-Fn-I (2DK-) with cys tail (SEQ ID NO: 53)

V-GS5-I (2DK-) with ser or cys tail (SEQ ID NO: 54)

V-GS10-I (2DK-) with ser or cys tail (SEQ ID NO: 55)

VI (DK +) (SEQ ID NO: 56)

VI (DK-) (SEQ ID NO: 57)

  The invention will now be described by reference to the following examples, which are illustrative only and are not intended to limit the invention. Although the invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made thereto without departing from the spirit and scope thereof. I will.

Fibronectin based scaffold proteins Generate a variety of fibronectin based scaffold proteins, including VEGFR2 / IGF-IR binder ("V / I binder") and EGFR / IGF-IR binder ("E / I binder") did. The following table represents the constructs described herein and their corresponding SEQ ID NOs.

Table 1. Overview of various V / I and E / I binders.

  SEQ ID NO: 22 is the amino acid sequence of the V / I (DK +) bivalent construct first described in WO2009 / 142773. V / I (DK +) contains a fibronectin domain that binds to IGF-IR and VEGFR2. The IGF-IR binding fibronectin core has the sequence shown in SEQ ID NO: 2, and the VEGFR2 binding fibronectin core has the sequence shown in SEQ ID NO: 3. The two domains are linked by a polypeptide linker (SEQ ID NO: 7) derived from the amino acid sequence linking the first and second Fn3 domains in human fibronectin. The I-binding subunit of V / I (DK +) contains a C-terminal extension (C1) having the amino acid sequence of SEQ ID NO: 46, ie, contains a DK site. The V-binding subunit of V / I (DK +) contains a C-terminal extension (C2) having the amino acid sequence of SEQ ID NO: 47, ie, contains a DK site.

  SEQ ID NO: 23 is the amino acid sequence of the E / I (DK +) bivalent construct. E / I (DK +) contains a fibronectin domain that binds to IGF-IR and EGFR. The IGF-IR binding fibronectin core has the sequence shown in SEQ ID NO: 2, and the EGFR2 binding fibronectin core has the sequence shown in SEQ ID NO: 39. The two domains are linked by a glycine-serine polypeptide linker having SEQ ID NO: 9. The I-binding subunit of E / I (DK +) contains a C-terminal extension (C1) having the amino acid sequence of SEQ ID NO: 46, ie, contains a DK site. The E-binding subunit of E / I (DK +) contains a C-terminal extension (C2) having the amino acid sequence of SEQ ID NO: 47, ie, contains a DK site.

  SEQ ID NO: 24 is the amino acid sequence of the E / I (DK-, no C-terminal) bivalent construct. E / I (DK-, no C-terminus) contains an IGF-IR core (SEQ ID NO: 2) and an EGFR core (SEQ ID NO: 39) linked by a glycine-serine linker (SEQ ID NO: 9). The IGF-IR binding subunit contains a C-terminal extension (C1) having the amino acid sequence of SEQ ID NO: 4, ie, contains an EK site rather than a DK site. The EGFR binding subunit contains a C-terminal extension (C2) having the amino acid sequence of SEQ ID NO: 5, ie, lacks a DK site.

  SEQ ID NO: 25 is the amino acid sequence of the E / I (2DK−) bivalent construct. E / I (2DK-) comprises an IGF-IR core (SEQ ID NO: 2) and an EGFR core (SEQ ID NO: 39) linked by a glycine-serine linker (SEQ ID NO: 9). The IGF-IR binding subunit contains a C-terminal extension (C1) having the amino acid sequence of SEQ ID NO: 4, ie, contains an EK site rather than a DK site. The EGFR binding subunit contains a C-terminal extension (C2) having the amino acid sequence of SEQ ID NO: 6, ie, contains an EK site rather than a DK site.

Expression of fibronectin based scaffold protein and expression of purified E / I molecule The E / I bivalent construct was expressed in soluble form in E. coli cells. Inclusion bodies are recovered by cell disruption and centrifugation. E / I protein is filtered and captured using column chromatography. The purified protein is then covalently attached to PEG via a maleimide chemistry with a single cysteine residue. The PEGylated product is then finished using column chromatography and formulated using tangential flow filtration.

Expression of the V / I molecule For expression of the V / I bivalent construct, the nucleotide sequence encoding the construct was cloned into an inducible expression vector and expressed in intracellular inclusions in E. coli cells. Cell bank vials generated from single sown colonies were used to inoculate shake flask cultures as inoculum for large scale fermentations. Alternatively, seed fermentation is used for inoculum culture depending on the final fermentation volume. Large-scale fermentation includes a growth phase to accumulate biomass and a production phase to produce a fibronectin based scaffold protein. For primary recovery, intracellular inclusion bodies were released from the recovered cells using a microfluidizer, recovered by centrifugation, and subsequently washed with buffer and water.

  The purification step for the bivalent construct uses resolubilization of inclusion bodies based on guanidine hydrochloride followed by protein refolding. The refolded protein is filtered and loaded onto a cation exchange chromatography column. The product is then purified using a hydrophobic interaction column and the resulting elution pool is PEGylated by the addition of PEG reagent to produce a PEGylated protein.

  The PEGylated protein is then purified across a second cation exchange chromatography column. The elution is concentrated to the target protein concentration and then exchanged into formulation buffer using ultrafiltration / diafiltration (UF / DF). The UF / DF product is filtered using a 0.22 μm final filter. The filtered product is then filled into vials to produce the final formulation.

Effect of protein concentration on V / I protein stability Effect of protein concentration on physical (aggregation) and chemical (fragmentation) stability of purified V / I (DK +) (SEQ ID NO: 22) Was tested. V / I (DK +) protein was formulated in 10 mM succinic acid, 5% sorbitol at pH 5.5. The V / I protein concentration was either 3 mg / mL or 5 mg / ml. Samples were stored for 12 months at 4 ° C., and the samples were collected and analyzed at 1st, 6th, 2nd, 3rd, 6th, 9th and 12th months.

  The amount of aggregation is measured by evaluating the percentage of total protein that has formed aggregates (measured as high molecular weight ("HMW") species) over time. Aggregation was measured using size exclusion-high performance liquid chromatography (SE-HPLC) analysis to assess the level of HMW over time. SE-HPLC analysis was performed using a Superdex 200 10/300 GL column with a mobile phase of 0.2 M potassium phosphate, 0.15 M sodium chloride, 0.02% sodium azide, pH 6.8. . With detection at 280 nm, the flow rate was 0.5 mL / min. The effect of protein concentration on V / I protein aggregation over time (0-12 months) is represented in FIG. V / I (DK +) aggregates at a rate of 0.3% / month at a concentration of 3 mg / mL. A higher protein concentration (5 mg / mL) leads to a faster aggregation rate.

  Fragmentation is measured by evaluating the percentage of total protein fragmented or “clipped” over time. The level of clipped protein was determined by utilizing reverse phase-high performance liquid chromatography (RP-HPLC). RP-HPLC was performed using a Varian PLRP-S column (4.6 * 250 mm, pore size 300 mm, particle size 5 μm). The separation of the various species is achieved via a gradient consisting of water / acetonitrile / trifluoroacetic acid. The flow rate was 1.0 mL / min. Double detection was performed at 280 nm (for protein related species) and with evaporative light scattering (for ELS, PEG related species). FIG. 2 shows that V / I (DK +) fragmentation was found to be less dependent on protein concentration than aggregation. The fragmentation rate for V / I (DK +) was ˜0.1% / month at 4 ° C.

  Based on these aggregation and fragmentation data, a formulation of V / I (DK +) with a protein concentration of 3 mg / mL is 5 mg / mL to minimize aggregation and ensure sufficient stability for one year. A concentration of mL would be preferred.

Effect of pH on V / I protein stability The effect of pH on the physical and chemical stability of purified V / I (DK +) (SEQ ID NO: 22) was tested. V / I (DK +) protein is formulated in 50 mM sodium chloride with a buffer composition that is 20 mM sodium acetate (for pH 4 and 5) or 20 mM sodium phosphate (for pH 6 and 7) and stored at 25 ° C. did.

  Samples were collected once a week for a period of 4 weeks and aggregation assessments were performed using SE-HPLC as described in Example 3. The effect of pH on V / I (DK +) protein aggregation over time (0-4 weeks) is represented in FIG. The lowest aggregation rate was observed in the sample with the lowest pH tested (pH 4.0). The highest aggregation rate was observed in the sample with the highest pH tested (pH 7.0).

  Samples were collected once a week for a period of 4 weeks and fragmentation assessments were performed using SE-HPLC as described in Example 3. The effect of pH on V / I (DK +) protein fragmentation is represented in FIG. Low pH (pH 4.0) was found to inhibit over time aggregation of V / I (DK +) protein (FIG. 3), while low pH was a protein fragment of V / I (DK +) protein. It was found to cause an increase in crystallization (Figure 4).

  Liquid chromatograph-mass spectrometry (LC-MS) was performed to identify clip sites in the fragmented V / I (DK +) protein. V / I (DK +) protein was formulated in pH 5.5, 10 mM sodium acetate, 150 mM sodium chloride at a protein concentration of 5 mg / ml. LC-MS was performed according to the RP-HPLC method described in Example 3 and subsequently coupled to a Thermo LTQ ion trap mass spectrometer (MS). The HPLC elution was split 1: 5 at 0.2 mL / min flow and directed to MS. Online detection at 280 nm was continued. FIG. 5 presents LC-MS data from this experiment, demonstrating that D95 and D200, the major sites where fragmentation occurs, and several aspartic acid (D) residues are involved in fragmentation. It should be noted that there are several D residues throughout the V / I (DK +) protein (eg D5, D9, D110), but only D95 and D200 are immediately followed by lysine residues. “VI des Met” indicates a cleavage event that occurs at the maleimide bond of the PEG conjugation as a result of thermal stress.

  Based on these experiments, aggregation and fragmentation are best balanced by preparing the formulation at pH 5.5, however, depending on only this pH level, either degradation pathway ( Aggregation and fragmentation) cannot be removed.

Assessing aggregation and fragmentation with E / I proteins The problem of aggregation and fragmentation instability observed with V / I (DK +) (SEQ ID NO: 22) is another structurally related bivalent construction. In order to confirm that it was a problem common to the body, the aggregation and fragmentation properties of E / I (DK +) (SEQ ID NO: 23) were also evaluated at several pH levels.

  E / I (DK +) was formulated in 10 mM succinic acid, 5% sorbitol at pH 4.0, 4.5 or 5.5. E / I (DK +) was formulated into the desired formulation by tangential flow filtration (TFF) using a 30 kD MWCO membrane. At least 6 dia-volume buffers were changed to achieve the final formulation. The resulting protein concentration was confirmed by A280 and adjusted to 5 mg / mL with additional formulation buffer. The formulated E / I (DK +) was sterile filtered through a laminar flow hood and filled into sterile glass vials for stability monitoring. The vial was capped and crimped and then placed in an incubator temperature controlled at 4, 25 and 37 ° C.

  The aggregation rate of E / I (DK +) was evaluated by performing SE-HPLC analysis. SE-HPLC uses a Shodex KW404-4F HPLC column (4.6 * 250 mm, pore size 300 mm, particle size 5 μm) and a mobile phase consisting of 10 mM succinic acid / 3% sorbitol / 0.4 M arginine at pH 5.5. It has been executed. The flow rate was 0.35 mL / min and detection was performed at 280 nm. FIG. 6 shows that, similar to V / I (DK +), the aggregation rate of E / I (DK +) is stored at a higher pH level at 25 ° C. than for samples stored at a lower pH at 25 ° C. Higher for the samples. The same data trend was also observed for the sample stressed at 37 ° C.

The effect of protein concentration on the aggregation rate of E / I (DK +) was also evaluated. Similar to V / I (DK +), Table 2 shows that after 4 weeks, higher concentrations (7 mg / ml) of E / I (DK +) have a higher percentage of aggregate formation (lower percentage of monomers). It is related to Table 2 demonstrates that reducing the pH with protein concentration further reduces the percentage of aggregation.

  E / I (DK +) fragmentation was assessed by performing RP-HPLC analysis as described in Example 3. FIG. 7 shows that, similar to V / I (DK +), it was higher for samples stored at a lower pH level at 25 ° C. than for samples stored at a higher pH at 25 ° C. The same data trend was also observed for the sample stressed at 37 ° C.

  LC-MS was performed as described in Example 4 to identify clip sites in the fragmented E / I (DK +) protein. E / I (DK +) protein was formulated in pH 4.0, 10 mM succinic acid, 5% sorbitol at a protein concentration of 5 mg / ml and maintained at 25 ° C. to induce protein stress. FIG. 8 presents LC-MS data from this experiment, with several aspartic acid (D) residues involved in fragmentation, including D95 and D218 (positions homologous to D200 in V / I (DK +)) Demonstrate that Fragmentation was also observed at D199. Since the D199 site is located in the FG binding loop of the EGFR binding region, it is unique to these E / I molecules and is not found in V / I molecules.

Evaluation of aggregation and fragmentation with DK-minus E / I variants Various E / I binders (SEQ ID NO: 23-25) were formulated and their physical (aggregation) and chemical (fragmentation) stability Were compared to each other under identical conditions (see Table 1). Based on the characterization of the D95 and D218 clip sites observed with E / I (DK +) (Example 5) and based on the fact that these DK clip sites are located in a structurally non-essential C-terminal tail, C Two different E / I constructs were generated in which the terminal tail DK site was removed or replaced.

  The E / I (DK +) molecule (SEQ ID NO: 23) is a control E / I binder containing a C-terminal tail containing a DK site (D95 and D218) after each of the two binding domains. The physical and chemical stability of this molecule was characterized in Example 5. The E / I (DK-, no C-terminus) molecule (SEQ ID NO: 24) does not contain any DK sites. In this molecule, the aspartate at position 95 is mutated to glutamic acid and the EIDKPCQ tail (SEQ ID NO: 47) is exchanged for the EGSGC tail (SEQ ID NO: 5). The E / I (2DK-) molecule (SEQ ID NO: 25) also does not contain any DK sites. In this molecule, aspartate at positions 95 and 218 has been exchanged for glutamic acid. V / I (DK +) was included in this experiment as a control.

  E / I protein (SEQ ID NO: 23-25) was formulated in 10 mM succinic acid, 5% sorbitol at pH 4.0. In addition, E / I (DK +) (SEQ ID NO: 23) and V / I (DK +) (SEQ ID NO: 22) were formulated in 10 mM succinic acid, 5% sorbitol at pH 5.5. Based on preliminary surfactant screening, surfactant was not found to be necessary. E / I (DK +) was formulated into the desired formulation by tangential flow filtration (TFF) using a 30 kD MWCO membrane. At least 6 dia-volume buffers were changed to achieve the final formulation. The resulting protein concentration was confirmed by A280 and adjusted to 5 mg / mL with additional formulation buffer. Each formulated bivalent construct was sterile filtered through a laminar flow hood and filled into sterile glass vials for stability monitoring. The vial was capped and crimped and then placed in an incubator temperature controlled at 4, 25 and 37 ° C.

  Aggregation rates of different E / I molecules were evaluated by performing SE-HPLC as described by Example 5, and the results from this experiment are illustrated in FIG. As expected, the rate of aggregation is significantly higher at pH 5.5 than at pH 4.0 for E / I (DK +) at 25 ° C. Based on the slope seen in FIG. 9, the aggregation rate for E / I (DK +) is approximately 7 times higher at 25.degree. C. than at pH 4.0. For the two E / I molecules lacking the DK site, the aggregation rate was not affected at pH 4.0. E / I (DK +) and V / I (DK +) exhibited similar aggregation rates at pH 5.5.

  Fragmentation rates of different E / I molecules were evaluated by performing RP-HPLC analysis as described in Example 3, and the results from this experiment are illustrated in FIG. With respect to clipping, FIG. 10 shows a lower clip rate at pH 5.5 than pH 4.0 for E / I (DK +) at 25 ° C. Among the molecules shown in FIG. 10, the highest clip rate was seen in E / I (DK +) at pH 4.0, which was significantly minimized when the pH of the formulation was increased to pH 5.5. However, as described above, this molecule had the highest aggregation rate at pH 5.5. For the other two E / I molecules lacking the DK site, the clipping rate at pH 4.0 was reduced approximately 3-fold compared to E / I (DK +) at the same pH. V / I (DK +) shows a higher degree of fragmentation compared to E / I (DK +) at the same pH (pK5.5), with V / I (DK +) and E / I (DK +) molecules Although similar, it shows that V / I (DK +) molecules are more susceptible to fragmentation.

  Characterization of the clipping site for E / I (DK−, no C-terminus) compared to the clipping site for E / I (DK +) using LC-MS as described in Example 4 It has been executed. Two different E / I binders were formulated in pH 4.0, 10 mM succinic acid, 5% sorbitol, respectively, at a protein concentration of 5 mg / ml and maintained at 25 ° C. to induce protein stress. FIG. 11 represents the LC-MS data from this experiment and demonstrates that the fragmentation profile is different between the two different E / I binders. The major aspartic acid (D) residues involved in fragmentation in E / I (DK +) were D95, D218 and D199. On the other hand, the main aspartic acid residues involved in fragmentation at E / I (DK-, no C-terminus) were D199, D82 and D193. However, as illustrated in Table 3 below, the percentage of total clips at E / I after 4 weeks (DK−, no C-terminus) is compared to E / I after this same period (DK +). Halved (3.8% compared to 7.5%). These results indicate that E / I (DK−, no C-terminus) is associated with reduced fragmentation compared to E / I (DK +).

  Characterization of the clipping site for E / I (2DK-) was performed using LC-MS as described in Example 4. E / I (2DK−) was formulated in pH 4.0, 10 mM succinic acid, 5% sorbitol at a protein concentration of 5 mg / ml and maintained at 25 ° C. to induce protein stress. FIG. 12 presents the LC-MS data from this experiment, with the major aspartic acid residues involved in fragmentation in E / I (2DK−) as well as E / I (DK−, no C-terminal). , D199, D82 and D193. Also, as illustrated in Table 3 below, the percentage of total clips in E / I (2DK−) after 4 weeks is more than half compared to E / I (DK +) after this same period. there were. These results indicate that E / I (2DK-) is associated with reduced fragmentation compared to E / I (DK +). .

Table 3. Amount and location of fragmentation for various E / I binders after 4 weeks of storage at 25 ° C.

  As described above, the D199 site is located within the FG binding loop of the EGFR binding region. Therefore, D199 is probably necessary for the binding function and will be difficult to remove from E / I (2DK-) or E / I (DK-, no C-terminal) molecules.

  The exact mechanism of clipping at the aspartic acid site observed with V / I (DK +) and E / I (DK +) is not clear. Based on the apparent pKa values of three aspartic acids at glucagon as measured by NMR methods, Joshi et al. (Journal of Pharmaceutical Sciences, 94 (9), 2005) Proposed mechanism. While not being bound by theory, some of the proposed mechanisms include the cyclization of aspartate side chains to form a five-membered ring, followed by peptide bond cleavage, which then causes peptide bond cleavage. May include nucleophilic attacks. By substituting aspartic acid with glutamic acid, ring formation does not occur easily due to steric hindrance, and thus may inhibit peptide bond cleavage at that position.

Assessment of aggregation and fragmentation in DK minus V / I variants The stability of two VEGFR-IGFR (VI) fibronectin based scaffold proteins was compared. The first construct is VI (DK +) (SEQ ID NO: 56) and the second construct VI (DK−) (SEQ ID NO: 57) is an asparagine at positions 94 and 199 of SEQ ID NO: 56. Contains substitution of acid with glutamic acid.

  Both molecules were formulated at a protein concentration of 3 mg / ml in 10 mM succinic acid, 5% sorbitol at pH 4.0, 4.5 and 5.5. The limited stability of these formulations was performed at 4 and 25 ° C. for up to 2 months, with periodic time points drawn for analysis by SE-HPLC and RP-HPLC. In addition, LC-MS characterization was performed on 2 month 25 ° C samples to determine the exact clip site on both proteins.

Effect of pH on aggregation rate in VI fibronectin based scaffold proteins Based on past experience with fibronectin based scaffold proteins, low pH formulations have the best biophysical stability for these molecules (Ie, lower aggregation) is recognized to provide. In this study, the two VI constructs demonstrate the same trend as seen previously, as illustrated in FIG. Although the onset level of aggregation is slightly different between the two molecules, the rates observed over the stability period are very similar at each given pH, and the aggregation rates for both molecules are in the same order, pH 5. 5 >> pH 4.5> pH 4.0.

Effect of pH on clip rate in VI fibronectin-based scaffold proteins Based on past experience with fibronectin-based scaffold proteins, if the protein sequence contains sites that are susceptible to clipping, low pH formulations It has been recognized that it provides the lowest chemical stability for these proteins. In past stability studies performed on VI (DK +) (SEQ ID NO: 56), a number of clip sites were identified and had the most severe clipping at D94 and D199. In this study, the two VI constructs demonstrated the same trend as previously seen, as illustrated in FIG. 14, and were worse for VI (DK +) than for VI (DK-) , Has an effect of pH on the clip rate. The clip rate follows the general trend of pH 4.0> pH 4.5> pH 5.5 for both molecules, but VI (DK−) is almost different in its clip rate over all three pH values. Indicates that there is no.

  From the stability data trends shown in FIGS. 13 and 14, when the formulation pH is reduced from 5.5 to 4.0, the clip rate per week for VI (DK +) increases 3.3-fold, while Now, for the VI (DK-) molecule, removal of the main clip site at two positions increased this rate by a factor of 1.6. Therefore, with these amino acid substitutions, the clip rate in VI fibronectin based scaffold proteins was reduced by approximately 50% over the same stability period when using the pH 4 formulation. On the other hand, when the formulation pH is reduced from 5.5 to 4.0, the aggregation rate per week for VI (DK +) and VI (DK−) decreases by 86 and 216 times, respectively. The pH effect on aggregation rate is therefore more dramatic than the effect on clip rate in VI fibronectin based scaffold proteins.

Identification of clip sites by LC-MS The structural characterization of clip sites observed in VI (DK +) and VI (DK-) was performed by LC-MS. An overlaid RP-HPLC chromatogram of the clip region from the 25 ° C./2 month time point is seen in FIG. 15 and peak identification is summarized in Table 4.

Table 4. Summary of peak identification by mass spectrometry. Residues highlighted in red indicate aspartic acid present in the original VI sequence that was exchanged for glutamic acid in the VI (DK-) construct.

  While some clips remain the same between the two molecules, the major clip sites at D94 and D199 in VI (DK-) were removed, and after 2 months stress at 25 ° C, the level of total clips Up to 6.9%. On the other hand, the total clip in VI (DK +) remained high at 16.0% over the same stability period. Low level of other clips were also identified in VI (DK−) but not in VI (DK +).

Table 5. Comparison of clip rate with VI (DK +) and VI (DK−).

  The clip rate of VI (DK +) at pH 4.0 was 1.6 times and 3.3 times faster than the clip rate of VI (DK +) at pH 4.5 and 5.5, respectively. The clip rate of VI (DK−) at pH 4.0 was 1.5 and 1.6 times faster than the clip rate of VI (DK−) at pH 4.5 and 5.5, respectively.

Table 6. Comparison of aggregation rates with VI (DK +) and VI (DK−).

  The aggregation rate of VI (DK +) at pH 4.5 and 5.5 was 4.4 times and 86 times faster than the aggregation rate of VI (DK +) at pH 4.0, respectively. The aggregation rate of VI (DK−) at pH 4.5 and 5.5 was 4.0 times and 216 times faster than the aggregation rate of VI (DK−) at pH 4.0, respectively.

  The stability results obtained with the two types of VI fibronectin based scaffold proteins show that selective replacement of problematic aspartic acid with glutamic acid in order to eliminate specific chemical degradation in the fibronectin based scaffold protein. Demonstrate the effectiveness and benefits of. With the major chemical degradation removed through this approach, better biophysical stability in fibronectin based scaffold proteins can now be achieved through formulation at low pH. Results from this study at VI, as well as previously obtained from EI dual functional fibronectin based scaffold proteins, are necessary to remove certain aspartic acids known to be prone to clipping In order to demonstrate sex and similarly maximize biophysical stability in fibronectin based scaffold proteins, formulation at acidic pH can be used.

Materials and Methods Formulation: Each molecule was formulated into the desired formulation via tangential flow filtration (TFF) using a 30 kD MWCO membrane. At least 6 dia-volume buffers were changed to achieve the final formulation. The resulting protein concentration was confirmed by A280 and adjusted to 3 mg / mL with additional formulation buffer.

  Vial Filling and Stability: Each formulated protein was sterile filtered through a laminar flow hood and filled into sterile glass vials for stability monitoring. The vial was capped and crimped and then placed in an incubator temperature controlled at 4 and 25 ° C.

analysis method:
Size-exclusion HPLC (SE-HPLC): Analysis was performed on a Shodex KW404-4F HPLC column (4.6 * 250 mm, pore size 300 mm, particle size 5 μm) and 10 mM succinic acid / 3% sorbitol / 0.4 M arginine at pH 5.5. Was carried out using a mobile phase consisting of The flow rate was 0.35 mL / min and detection was performed at 280 nm.

  Reversed phase HPLC (RP-HPLB): The analysis was performed using a Varian PLRP-S column (4.6 * 250 mm, pore size 300 mm, particle size 5 μm). The separation of the various species is achieved via a gradient consisting of water / acetonitrile / trifluoroacetic acid. The flow rate was 1.0 mL / min. Double detection was performed at 280 nm (for protein related species) and with evaporative light scattering (for ELS, PEG related species).

  LC-MS: Clip site characterization was performed using a Jupiter C18 column (4.6 * 250 mm, pore size 300 mm, particle size 5 μm) coupled to Thermo LTQ ion trap mass spectrometry (MS). The HPLC elution was split 1: 5 at 0.2 mL / min flow and directed to MS. Online detection at 280 nm was continued.

Incorporation by explicit reference It is individually incorporated into this document.

Claims (46)

A polypeptide comprising a fibronectin type III No. ¹⁰ (10 Fn3) domain, the ¹⁰ Fn3 domain, SEQ ID NO: 1 and comprises an amino acid sequence having at least 60% identity, to a target molecule with a _{K D} of less than 100nM A polypeptide that binds and wherein the ¹⁰ Fn3 domain further comprises a C-terminal tail that does not contain a DK sequence.   The polypeptide of claim 1, wherein the C-terminal tail comprises the amino acid sequence of SEQ ID NO: 4.   The polypeptide of claim 1 or 2, wherein the C-terminal tail further comprises a cysteine residue.   4. The polypeptide of claim 3, wherein the C-terminal tail comprises the amino acid sequence of SEQ ID NO: 5. The polypeptide according to any one of claims 1-4, wherein the ¹⁰ Fn3 domain further comprises an N-terminal extension comprising 1-10 amino acids.   6. The polypeptide of claim 5, wherein the N-terminal extension comprises a sequence selected from the group consisting of M, MG, G and any one of SEQ ID NOs: 19-21 and 26-31. Further comprising a second ¹⁰ Fn3 domain, a polypeptide according to any one of claims 1-6, wherein the second ¹⁰ Fn3 domain, SEQ ID NO: having one at least 60% identity comprises an amino acid sequence, it binds to the target molecule with a _{K D} of less than 100 nM, and, said second ¹⁰ Fn3 domain further comprises a C-terminal tail containing no DK sequence polypeptide. 8. The polypeptide of claim 7, wherein the second ¹⁰ Fn3 domain comprises a C-terminal tail comprising the amino acid sequence of SEQ ID NO: 4. 9. A polypeptide according to claim 7 or 8, wherein the first and second ¹⁰ Fn3 domains bind to different targets. ^10. The polypeptide of any one of claims 7-9, wherein the second ¹⁰ Fn3 domain further comprises an N-terminal extension comprising 1-10 amino acids.   12. The polypeptide of claim 10, wherein the N-terminal extension comprises a sequence selected from the group consisting of SEQ ID NOs: 19-21 and 26-31. 114. The polypeptide of any one of claims 7-11, wherein the first and second ¹⁰ Fn3 domains are linked by a polypeptide linker comprising 1-30 amino acids.   13. The polypeptide of claim 12, wherein the polypeptide linker is selected from the group consisting of a glycine-serine based linker, a glycine-proline based linker, a proline-alanine linker and an Fn based linker.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 48. A polypeptide comprising an amino acid sequence having an N1-D1-C1-L-N2-D2-C2 structure,
N1 and N2 are any N-terminal extensions independently containing 0-10 amino acids;
D1 and D2, (i) SEQ ID NO: 10 fibronectin type III domain having 2 amino acid sequence shown in at least 95% identity ⁽¹⁰ Fn3), here, the ¹⁰ Fn3 domain with a _{K D} of less than 500nM one that binds to IGF-IR, and (ii) SEQ ID NO: ¹⁰ Fn3 domain having an amino acid sequence at least 95% identity with shown in 3, where said ¹⁰ Fn3 domain with a _{K D} of less than 500 nM VEGFR2 Independently selected from the group consisting of:
L is a polypeptide linker comprising 0-30 amino acid residues;
A polypeptide wherein C1 comprises the amino acid sequence of SEQ ID NO: 4; and C2 comprises the amino acid sequence of SEQ ID NO: 4, 5 or 6. D1 is a ¹⁰ Fn3 domain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2, and D2 is a ¹⁰ Fn3 domain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 3 16. The polypeptide of claim 15, wherein D1 is a ¹⁰ Fn3 domain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 3, and D2 is a ¹⁰ Fn3 domain having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2. 16. The polypeptide of claim 15, wherein   18. The polypeptide according to any one of claims 15-17, wherein C2 comprises the amino acid sequence of SEQ ID NO: 6.   19. A linker according to any of claims 15-18, wherein L is a linker selected from the group consisting of a glycine-serine based linker, a glycine-proline based linker, a proline-alanine linker and an Fn based linker. The polypeptide according to claim 1.   20. The polypeptide of claim 19, wherein L comprises the amino acid sequence of SEQ ID NO: 7.   21. The polypeptide of any one of claims 15-20, wherein N1 comprises an amino acid sequence selected from the group consisting of M, MG, G and any one of SEQ ID NOs: 19-21 and 26-31.   The polypeptide of claim 21, wherein N1 comprises the amino acid sequence of SEQ ID NO: 19.   23. The poly of any one of claims 15-22, wherein N2 comprises an amino acid sequence selected from the group consisting of M, MG, G and any one of SEQ ID NOs: 19-21 and 26-31. peptide.   24. The polypeptide of claim 23, wherein N2 comprises the amino acid sequence of SEQ ID NO: 20.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 25.   26. The polypeptide of any one of claims 1-25, wherein the polypeptide has a fragmentation of less than 4% during storage in solution at pH 4.0 for at least 4 weeks.   27. The method of any one of claims 1-26, further comprising one or more pharmacokinetic (PK) moieties selected from a polyoxyalkylene moiety, a human serum albumin binding protein, sialic acid, human serum albumin, transferrin and an Fc fragment. The described polypeptide.   28. The polypeptide of claim 27, wherein the PK moiety is a polyoxyalkylene moiety and the polyoxyalkylene moiety is polyethylene glycol.   A pharmaceutical composition comprising the polypeptide according to any one of claims 1-28.   30. The pharmaceutical composition according to claim 29, further comprising a physiologically acceptable carrier.   31. A pharmaceutical composition according to claim 29 or 30, wherein the composition is essentially pyrogen-free.   32. The pharmaceutical composition according to any one of claims 29-31, wherein the pH of the composition is between 4.0-6.5.   34. The pharmaceutical composition according to claim 32, wherein the pH of the composition is between 4.0-5.5.   32. The pharmaceutical composition according to any one of claims 29-31, wherein the pH of the composition is 4.0.   35. The pharmaceutical composition according to any one of claims 29-34, wherein the polypeptide concentration is at least 5 mg / mL. 29. A pharmaceutical formulation comprising the polypeptide of any one of claims 1-28, wherein the formulation comprises at least 5 mg / mL polypeptide, has a pH of 4.0, and is administered intravenously. Suitable for pharmaceutical preparations.   37. A pharmaceutical formulation according to claim 36, wherein the formulation is stable at 25 [deg.] C for at least 4 weeks.   38. The pharmaceutical formulation of claim 37, wherein the formulation has a fragmentation of less than 4%.   39. A pharmaceutical formulation according to claim 37 or 38, wherein the formulation has an aggregation of less than 4%.   40. A method for treating a hyperproliferative disorder in a subject comprising administering to the subject in need a therapeutically effective amount of a composition according to any one of claims 29-39.   A nucleic acid encoding the polypeptide according to any one of claims 1-26.   42. A vector comprising the nucleic acid of claim 41.   43. The vector of claim 42, wherein the vector is an expression vector.   44. A host cell comprising the vector of claim 43.   45. The host cell of claim 44, wherein the cell is a microbial cell.   46. The host cell according to claim 45, wherein the cell is a mammalian cell.

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